Circulating miRNAs are potential biomarkers of various pathological conditions¹ and have been characterized previously in total urine specimens and exosomes from body fluids other than urine², but have yet to be studied in urinary exosomes. Advances have been made in understanding the role of miRNAs in cancer pathogenesis, but less is known about their role in other chronic diseases. Studies have been reported associating certain miRNAs with hypertension but miRNAs have not yet been directly linked to sodium metabolism.

To characterize the urinary exosome miRNome, researchers at the University of Virginia Health System used LC Sciences microarrays to explore the miRNA spectrum present within urinary exosomes from ten individuals that had completed a salt sensitivity clinical study³. Groups exhibiting extremes of salt sensitivity of blood pressure were compared to a group of normal individuals who had blood pressure that did not change dependent on sodium consumption, i.e. they were salt-resistant. In the microarray, potential biomarkers were sought based on these three phenotypes defined below.

Of the 194 miRNAs detected, 45 were significantly different between the SS and SR individuals or between the ISS and SR subjects. A search of the literature for each of the 45 miRNAs that were significantly different between SS or ISS vs. SR individuals, revealed that only 21 were previously cited as extracellular secreted miRNAs in serum or other bodily fluids (http://atlas.dmi.unict.it/mirandola/index.html). The 24 miRNAs that have not been described as secreted from cells or found in body fluids can be considered unique to urinary exosomes and strongly indicate that serum exosomal miRNAs do not pass into the urine in appreciable quantities.

Investigating renal cellular pathophysiology through the study of intracellular biomarkers has the potential of providing a deeper understanding of how individuals express unique patterns of salt sensitivity of blood pressure. Ultimately, personalized therapeutic approaches to controlling salt-related illnesses will result from these new diagnostic techniques.

1. M. Ciesla, K. Skrzypek, M. Kozakowska, A. Loboda, A. Jozkowicz, J. Dulak. (2011) MicroRNAs as biomarkers of disease onset. Anal Bioanal Chem 401,2051–2061. [abstract]
2. K.C. Miranda, D.T. Bond, M. McKee, J. Skog, T.G. Păunescu, N. Da Silva, D. Brown, L.M. Russo. (2010) Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 78, 191–199. [abstract]
3. Gildea JJ, Carlson JM, Schoeffel CD, Carey RM, Felder RA. (2013) Urinary Exosome miRNome Analysis and its Applications to Salt Sensitivity of Blood Pressure. Clin Biochem [Epub ahead of print]. [abstract]

MicroRNAs (miRNAs), endogenous regulators of many genes, are a class of small noncoding RNAs that mediate post-transcriptional gene silencing. Researchers at Kyungpook National University, Korea performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross-mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. They investigated in detail one miRNA, miR-24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs.

• Choi JW, Kang SM, Lee Y, Hong SH, Sanek NA, Young WS, Lee HJ. (2013) MicroRNA profiling in the mouse hypothalamus reveals oxytocin-regulating microRNA. J Neurochem [Epub ahead of print]. [abstract]

Friday June 14th, 2013
Rush University Medical Center, The SEARLE Conference Center
1725 W Harrison St., Professional Building, Chicago, IL

You can download a copy of the slides with links here

MicroRNAs have proven to be an extremely important part of the gene expression regulation mechanism.  While the role of microRNAs in cancer was recognized early on, expression profiling and functional studies have now shown that microRNAs participate in the regulation of many biological systems. There is now strong evidence suggesting that miRNA signaling plays a significant role in both innate and adaptive immune responses and their dysregulation contributes to pathogenesis.

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments of microRNA research in the field of immunology
• Current methods for microRNA discovery and profiling
• Case studies and application examples

Now, researchers from the Chinese Academy of Medical Sciences and Peking Union Medical College studied microRNAs in Hep11 and Hep12 cells derived from primary and recurrence (intrahepatic metastatic) sites of hepatocellular carcinoma (HCC), respectively.

They found that Hep12 exhibited a higher invasive and migratory potential than Hep11. There was also a significantly higher expression of miR-9 in Hep12 cells than in Hep11 cells. Further studies in HCC cell lines demonstrated that miR-9 could promote tumor cell migration and invasion. In addition, miR-9 downregulated KLF17 protein expression by targeting the 3′UTR region of the KLF17 gene directly. As a transcription factor, KLF17 directly acted on the promoters of EMT-related genes (ZO-1, Vimentin and Fibronectin (FN)) in HCC cell lines.

Therefore, the authors conclude that miR-9 may possibly promote HCC migration and invasion through regulation of KLF17.

• Suna Z, Hanb Q, Zhoua N, Wanga S, Luc S, Baia C,  Zhaob C. (2013) MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma. Molecular Oncology [Epub ahead of print]. [abstract]

Sun Z, Han Q, Zhou N, Wang S, Lu S, Bai C, Zhao RC. (2013) MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma. Molecular Oncology [Epub ahead of print] . [abstract]

Cheng K, Rai P, Plagov A, Lan X, Mathieson PW, Saleem MA, Husain M, Malhotra A, Singhal PC. (2013) Rapamycin-induced modulation of miRNA expression is associated with amelioration of HIV-associated nephropathy (HIVAN). Experimental Cell Research [Epub ahead of print].  [abstract]

Title: Recent Work & Current Methods in Immunology microRNA Research

Date: Tuesday – 05/0713

MicroRNAs have proven to be an extremely important part of the gene expression regulation mechanism.  While the role of microRNAs in cancer was recognized early on, expression profiling and functional studies have now shown that microRNAs participate in the regulation of many biological systems. There is now strong evidence suggesting that miRNA signaling plays a significant role in both innate and adaptive immune responses and their dysregulation contributes to pathogenesis.

Presented by
Dr. Christoph Eicken

Head of Technical Services

LC Sciences LLC

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments of microRNA research in the field of immunology
• Current methods for microRNA discovery and profiling
• Case studies and application examples

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America – 1:00 Central Standard Time
https://attendee.gotowebinar.com/register/4876096924603080192

Europe, Asia – 2:00 PM Greenwich Mean Time
https://attendee.gotowebinar.com/register/5157900655290357248

After registering, you will receive a confirmation email containing information about joining the webinar.

After completion of the webinar, you will receive an email containing a link to the archived webinar.

Researchers at the Nanjing Normal University, China used the Illumina/Solexa deep sequencing technology to sequence small RNA libraries prepared from the fourth-instar larvae of male and female E. sinensis. 19,097,799 raw reads were yielded in total: 7,817,445 reads from the female library and 11,280,354 from the male, respectively. As a result, they identified 168 known miRNAs belonging to 55 families as well as 204 novel miRNAs. Moreover, 45 miRNAs showed significantly different expression between the female and the male fourth-instar larvae, and they validated 10 of them by Stem-loop qRT-PCR. Some of these differentially expressed miRNAs are related to metamorphosis, development and phenotypic diversity.

This is the first comprehensive description of miRNAs in E. sinensis. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs. These findings not only enrich miRNAs for hemimetabolans but also lay the foundation for the study of post-transcriptional regulation on the phenomena of sexual dimorphism.

• Wu W, Ren Q, Li C, Wang Y, Sang M, et al. (2013) Characterization and Comparative Profiling of MicroRNAs in a Sexual Dimorphism Insect, Eupolyphaga sinensis Walker. PLoS ONE 8(4), e59016. [article]

The differentiation of human peripheral blood monocytes into macrophages can be reproduced ex vivo by culturing the cells in the presence of Colony-Stimulating Factor 1 (CSF1). Using microarray profiling to explore the role of microRNAs (miRNAs), researchers from the French Institute of Health and Medical Research have identified a dramatic decrease in the expression of the hematopoietic specific miR-142-3p.

Up- and down-regulation of this miRNA in primary human monocytes altered CSF1-induced differentiation of monocytes, as demonstrated by changes in the expression of the cell surface markers CD16 and CD163. One of the genes whose expression is repressed by miR-142-3p encodes the transcription factor Early Growth Response 2 (Egr2). In turn, Egr2 associated with its co-repressor NGFI-A (Nerve Growth Factor-Induced gene-A) binding protein 2 (NAB2), binds to the pre-miR-142-3p promoter to negatively regulate its expression. Interestingly, the expression of miR-142-3p is abnormally low in monocytes from patients with the most proliferative forms of chronic myelomonocytic leukemia (CMML), and miR-142-3p re-expression in CMML dysplastic monocytes can improve their differentiation potential.

Altogether, miR-142-3p which functions in a molecular circuitry with Egr2, is an actor of CSF1-induced differentiation of human monocytes whose expression could be altered in CMML.

• Lagrange B, Martin RZ, Droin N, Aucagne R, Paggetti Jrm, Largeot A, Itzykson Rl, Solary E, Delva L, Bastie J-. (2013) A role for miR-142-3p in Colony-Stimulating Factor 1-induced monocyte differentiation into macrophages. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research [Epub ahead of print]. [abstract]

• CE Meacham. (2012) In vivo pool-based shRNA screens to identify modulators of disease progression in hematopoietic malignancies. MIT Thesis [abstract]

microRNA Sequencing

• Wu W, Ren Q, Li C, Wang Y, Sang M, Zhang Y, Li B. (2013) Characterization and Comparative Profiling of MicroRNAs in a Sexual Dimorphism Insect, Eupolyphaga sinensis Walker. PLoS ONE 8(4), [abstract]

• Ma J, Yu S, Wang F, Bai L, Xiao J, Jiang Y, Chen L, Wang J, Jiang A, Li M. (2013) MicroRNA Transcriptomes Relate Intermuscular Adipose Tissue to Metabolic Risk. International Journal of Molecular Sciences 14(4), 8611-8624. [abstract]

microRNA Microarray

• Murray AR, Chen Q, Takahashi Y, Zhou KK, Park K, Ma J. (2013) MicroRNA-200b Downregulates Oxidation Resistance 1 (Oxr1) Expression in the Retina of Type 1 Diabetes Model. Investigative ophthalmology & visual science 54(3), 1689-1697. [abstract]

• Ye H, Ling S, Castillo AC, Thomas B, Long B, Qian J, Perez-Polo JR, Ye Y, Chen X, Birnbaum Y. (2013) Nebivolol Induces Distinct Changes in Profibrosis MicroRNA Expression Compared With Atenolol, in Salt-Sensitive Hypertensive Rats. Hypertension [abstract]

• Liao L, Yang X, Su X, Hu C, Zhu X, Yang N, Chen X, Shi S, Shi S, Jin Y. (2013) Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow. Cell Death & Disease 4(4), [abstract]

• Lagrange B, Martin RZ, Droin N, Aucagne R, Paggetti Jrm, Largeot A, Itzykson Rl, Solary E, Delva L, Bastie J-. (2013) A role for miR-142-3p in Colony-Stimulating Factor 1-induced monocyte differentiation into macrophages. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research [abstract]

• May 14 -  Baylor College of Dentistry Biomedical Sciences;10:30 AM – 1:30 PM; Dallas, TX
• May 15 -  Univ of TX Arlington; 10:30 AM – 1:30 PM; Arlington, TX

These shows are being sponsored by The Genomics core at UT Arlington.

Bing Zhu¹, Ailing Hong², Chris Hebel², Xiaochuan Zhou² and Xiaolian Gao¹

¹ Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA.

² LC Sciences, Houston, TX 77054

Post-translational modifications (PTMs) of histones play a critical role in diverse biological processes including chromatin compaction, gene expression and cell differentiation. Among a myriad of PMTs, histone methylation catalyzed by histone methyltransferases (HMTs) has been increasingly recognized as an important player responsible for a major signaling mechanism in eukaryotic cells. This suite of epigenetic modifiers represents a new and promising class of therapeutic targets. In cancer, there is a growing body of evidence that suggests changes in the activity of HMTs (a class of chromatin-modifying enzymes) contribute to the uncontrolled cell proliferation that is a hallmark of this devastating disease. The sequence specificity of the substrates of HMTs under a cellular condition are largely unknown but known targets have been mostly identified through a conventional candidate-based approach by using purified HMTs. However, such an experiment frequently does not reflect what could be occurring in cellular contexts or in vivo. In this study, we designed and synthesized a comprehensive histone peptide microarray (PepArray) on a microfluidic chip which contains 3,919 peptides. The peptides contain nine residues with the methylation sites and mutant sites situated in the middle of the sequence so that each peptide has a unique possibility for modification such as methylation or acetylation. We obtained nuclear extract from the breast cancer cell line T47D, and applied the protein lysates to the histone methylation PepArray chip. After incubation of the chip with a methyl-specific antibody, significant signals were detected at the sites containing peptides corresponding to H2AK74, H3K122, and H4K59. We found null signal at mutant sites where the target lysine(K) was replaced with alanine(A). These results reveal the specific activity profiles of HMTs at defined histone sites in a cellular system. Planned further investigation will compare the different histone methylation or acetylation profiles in the various cellular systems, especially in different cancer systems. The current experiment demonstrates an effective solution to comprehensive studies of epigenetic modification. This information may be translated into therapeutic targets of histone methylation inhibition by focusing on identifying inhibitors of specific HMTs as targets for a new generation of therapeutics.

Two miRNA libraries were constructed from MG tissues taken from a lactating and a non-lactating Holstein dairy cow, respectively, and the short RNA sequences (18-30 nt) in these libraries were sequenced by Solexa sequencing method. The libraries included 885 pre-miRNAs encoding for 921 miRNAs, of which 884 miRNAs were unique sequences and 544 (61.5%) were expressed in both periods.

A custom-designed microarray assay was then performed to compare miRNA expression patterns in the MG of lactating and non-lactating dairy cows. A total of 56 miRNAs in the lactating MG showed significant differences in expression compared to non-lactating MG (P<0.05). Integrative miRNA target prediction and network analysis approaches were employed to construct an interaction network of lactation-related miRNAs and their putative targets. Using a cell-based model, six miRNAs (miR-125b, miR-141, miR-181a, miR-199b, miR-484 and miR-500) were studied to reveal their possible biological significance.

This study provides a broad view of the bovine MG miRNA expression profile characteristics. Eight hundred and eighty-four miRNAs were identified in bovine MG. Differences in types and expression levels of miRNAs were observed between lactating and non-lactating bovine MG. Systematic predictions aided in the identification of lactation-related miRNAs, providing insight into the types of miRNAs and their possible mechanisms in regulating lactation.

• Li Z, Liu H, Jin X, Lo L, Liu J. (2013) Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation. BMC Genomics 13, 731. [article]

Realizing the developmental and therapeutic potential of hESCs has been hindered by conventional multi-lineage differentiation of pluripotent cells, which is uncontrollable, inefficient, highly variable, difficult to reproduce and scale-up. We recently identified retinoic acid (RA) as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs under defined platform and trigger progression to human neuronal progenitors (hESC-I hNuPs) and neurons (hESC-I hNus) in the developing CNS with high efficiency, which enables hESC neuronal lineage-specific differentiation and opens the door to investigate human embryonic neurogenesis using the hESC model system.

In this study, genome-scale profiling of microRNA (miRNA) differential expression patterns in hESC neuronal lineage-specific progression was used to identify molecular signatures of human embryonic neurogenesis. These in vitro neuroectoderm-derived human neuronal cells have acquired a neuronal identity by down-regulating pluripotence-associated miRNAs and inducing the expression of miRNAs linked to regulating human CNS development to high levels in a stage-specific manner, including silencing of the prominent pluripotence-associated hsa-miR-302 family and drastic expression increases of the Hox hsa-miR-10 and let-7 miRNAs. Following transplantation, hESC-I hNuPs engrafted and yielded well-integrated neurons at a high prevalence within neurogenic regions of the brain.

This study provides critical insight into molecular neurogenesis in human embryonic development as well as offers an adequate human neurogenic cell source in high purity and large quantity for scale-up CNS regeneration.

• Parsons XH, Parsons JF, Moore DA. (2013) Genome-Scale Mapping of MicroRNA Signatures in Human Embryonic Stem Cell Neurogenesis. Mol Med Ther 1(2). [article]

Liu N, Yang J, Guo S, Xu Y, Zhang M. (2013) Genome-Wide Identification and Comparative Analysis of Conserved and Novel MicroRNAs in Grafted Watermelon by High-Throughput Sequencing. PloS one 8(2). [article]

Microarray

Wang W, Cheng B, Miao L, Mei Y, Wu M. (2013) Mutant p53-R273H gains new function in sustained activation of EGFR signaling via suppressing miR-27a expression. Cell Death & Disease 4(4). [abstract]

Tzarfati R, Ben-Dor S, Sela I, Goldschmidt EE. (2013) Graft-induced Changes in MicroRNA Expression Patterns in Citrus Leaf Petioles. The Open Plant Science Journal 7(1), 17-23. [article]

Cheng Y, Du L, Shi Q, Jiao H, Zhang X, Hao Y, Rong H, Zhang J, Jia X, Guo S. (2013) Identification of miR-221 and -222 as important regulators in genotype IV swine hepatitis E virus ORF3-expressing HEK 293 cells. Virus Genes 1-7. [abstract]

Chen D, Wang W. (2013) Human Placental MicroRNAs and Preeclampsia. Biology of Reproduction  [Epub ahead of print]. [abstract]

You’re invited to come by and discover the breath of services offered by LC Sciences to assist you in making breakthrough cancer research discoveries!

Meet our Team:

Chris Hebel, Vice President of Business Development
Dr. Qi Zhu, Senior Scientist
Jason Mulcahey, Account Manager, Genomics Division

Drop by our poster presentation:

Poster Session: Tues, 4/9/13 1:00PM – 5:00PM
Location: Hall A-C, Poster Session 32
Title: Site Specific Profiling of Histone Methyltransferases in Cancer Cells Using Histone Peptide Microarray Containing a Comprehensive Set of Histone Peptides
Presented by: Bing Zhu^, Department of Biology and Biochemistry, University of Houston, Houston, TX 77004

Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very similar to known miRNAs from other insects, and 184 novel miRNAs that have not been reported from other species. A genome-wide search through a draft Hessian fly genome sequence identified a total of 611 putative miRNA-encoding genes based on sequence similarity and the existence of a stem-loop structure for miRNA precursors. Analysis of the 611 putative genes revealed a striking feature: the dramatic expansion of several miRNA gene families. The largest family contained 91 genes that encoded 20 different miRNAs.

Microarray analyses revealed the expression of miRNA genes was strictly regulated during Hessian fly larval development and abundance of many miRNA genes were affected by host genotypes.

The identification of a large number of miRNAs for the first time from a gall midge provides a foundation for further studies of miRNA functions in gall midge biology and behavior. The dramatic expansion of identical or similar miRNAs provides a unique system to study functional relations among miRNA iso-genes as well as changes in sequence specificity due to small changes in miRNAs and in their mRNA targets. These results may also facilitate the identification of miRNA genes for potential pest control through transgenic approaches as was demonstrated on the cotton bollworm².

1. Khajuria C, Williams CE, Bouhssini ME, Whitworth RJ, Richards S, Stuart JJ, Chen M. (2013) Deep sequencing and genome-wide analysis reveals the expansion of MicroRNA genes in the gall midge Mayetiola destructor. BMC Genomics 14(1), 187. [article]
2. Jayachandran B, Hussain M, Asgari S. (2012) An insect trypsin-like serine protease as a target of miRNA: utilization of miRNA mimics and inhibitors by oral feeding. Insect Biochem Mol Biol [Epub ahead of print]. [abstract]

Note – We would like to thank the group at Kansas State University for generously allowing us to use this study as an application example of Seq-Array on our poster.

MicroRNA (miRNA) microarray analysis has consistently found altered expression of miRNAs in thyroid tumors, suggesting their roles in thyroid carcinogenesis and diagnosis. MiRNA is a non-coding small RNA existing extensively in plants, animals, and viruses, at an approximate length of 21-23 nt and highly conserved. It binds to specific mRNAs and regulates gene transcription.  Mature miRNAs form with other proteins into RNA induced silencing complex, resulting in the degradation or translation suppression of target mRNA if binding to target. miRNAs are involved in the regulation of multiple critical biological activities, included cell apoptosis, proliferation, and tumor genesis. The expression of miRNA is significantly differential between tumor and normal tissue, in addition to among various tumor tissues, suggesting the critical role of miRNAs in tumor genesis.

To explore the altered different expression of miRNA and the mechanisms underlying the pathogenesis of PTC, researchers at Zhengzhou University, China examined the differential expression profiles of miRNAs in stage I-III PTC by using miRNA microarray chip. They also made a preliminary analysis of biological function for the most differentially expressed miRNAs and their potentially regulated target genes.

Ten miRNAs exhibited sequential up regulation expression profiles and five miRNAs performed sequential down regulation throughout stage I to III (p<0.05). After normalization, Fifteen miRNAs showed significant different compared to adjacent non-tumor tissues (p<0.05). Among of them, the most significant up regulation and down regulation miRNAs were miR-146b-5p and miR-335, respectively. Both of them were verified with qRT-PCR. 34 target genes for miR-146-5p and 36 target genes for miR-335 was predicted.

MicroRNA microarray profiling successfully detected a branch of differential expression miRNAs between PTC and normal tissue. Some of them also were demonstrated to be stage specific. Biological function analysis showed that target genes were involved in five aspects including cell proliferation, differentiation, apoptosis, cycle, and signaling transduction pathway, suggesting the regulatory role of abnormal expression of critical miRNAs in the pathogenesis of PTC.

• Zhang J, Liu Y, Liu Z, Wang XM, Yin DT, Zheng LL, Zhang DY, Lu XB. (2013) Differential Expression Profiling and Functional Analysis of microRNAs through Stage I-III Papillary Thyroid Carcinoma. Int J Med Sci 10(5), 585-592. [article]

Fu HL, Wu DP, Wang XF, Wang JG, Jiao F, Song LL, Xie H, Wen XY, Shan HS, Du YX. (2013) Altered miRNA Expression is Associated with Differentiation, Invasion, and Metastasis of Esophageal Squamous Cell Carcinoma (ESCC) in Patients from Huaian, China. Cell Biochem Biophys [Epub ahead of print]. [article]

Download the presentation slides with reference links

Presenter Name: Dr. Qi Zhu -

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post-transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules (targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA-target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.

Recently, degradome sequencing, a modified 5′-rapid amplification of cDNA ends (RACE) performed with next-gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation. This presentation will provide an overview of this emerging method for miRNA target identification, as well as practical information for performing degradome sequencing experiments and data analysis. Diverse case study examples will be provided to illustrate the usefulness of this latest application of next-gen sequencing technology to plant miRNA research.

The time to start thinking about validating your microarray results is not after you have completed your experiment, but instead before you start. Generally, your ability to validate results is dependent on the statistical validity of your data which is in turn dependent on good experimental design.

Good experimental design is essential to ensure statistically significant results and to justify scientific conclusions. Good basic experimental design includes randomization of samples, (assignment to conditions, prep order, location in instrument, etc.) and the use of biological replicates. Biological variability is a fundamental characteristic of gene expression and for experiments performed with a small number of biological replicates, results may be due to biological variation and/or experimental variation rather than the difference in conditions/treatment. Hence, they may not be reproducible; and it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the different sample groups. Therefore, you will not be able to validate results from poorly designed experiments (e.g. experiments with only a single sample per test group)

The appropriate number of biological replicates to use is dependent on the specifics of your experiment (e.g. species, sample type, expected degree of variability, effect that treatment or condition has on miRNA expression).[1]

Validation Expectations

We would define a validated result as one that demonstrates a change in expression in the same direction (e.g. up or down) as the original result. The fold change or the degree of change in expression may differ simply due to the differences inherent to these methods of determining expression levels. For examples, TaqMan QPCR is a very specific method and measures the expression level of one very specific miRNA sequence. Microarray on the other hand, will not necessarily distinguish the difference between the potential isoform sequences of any given miRNA – e.g, if the iso-miRNA is just a shorter version or sequence shifted version. Additionally, TaqMan QPCR and array assays use different enzymatic reactions involving reverse transcription and ligation, respectively. The two reactions do not always have the same yield.

Sample Selection

It is very important to always use the exact same samples in validation experiments as you did in the original microarray experiment. You may not be able to validate expression results using similar samples or replicate samples from the same group, or even from a separate culture dish. There is natural variability in expression levels between individual specimens and experimental variability between RNA extractions, hence the need for replicates in experimental design. Therefore, the sample tested should come from the exact same RNA extraction prep as was used for the original microarray experiment.

miRNA Sequence Selection

There are three criteria that should be met in order to consider that a miRNA expression result can be validated: signal intensity, fold-change, and p-value.

     Signal Intensity Value

The signal intensity for each miRNA can be found in the normalized data file named S1XXXXX_MultiArray Analysis_Data.xls. You can use the following intensity values as a guideline to categorize your results:

< 500 represent very low intensity data

~500-2000 – low intensity

~2000-10,000 – medium intensity

> 10,000 – high intensity

For validation experiments, we recommend to focus on those miRNAs that show a minimum intensity of 500 in at least one of the sample groups.

Lower intensity signal miRNAs will be difficult to validate because the QPCR cycle numbers (and hence standard deviation) will be increased. In these cases, the standard deviation may become higher than the fold-change you are trying to validate.

     Fold Change

The fold-change value for each miRNA has been calculated and can be found in the far right column of the statistical test in your In-depth Data Analysis Report and are stated as the log₂ values.

We recommend selecting miRNAs for validation that exhibit at least a 2-fold change in expression which corresponds to a log₂ value of > +1 or <-1.

     p-Value

The p-value for each miRNA has been calculated and can be found in the statistical test result tables (e.g. T-test-01-XXXX.xls) in the second column – adjacent to the miRNA name. It is a measure of the statistical significance of a given expression result.

Unfortunately, there is no hard and fast p-value criteria for validation as this value will be dependent on the number of samples in your experiment. In general, a lower p-value is a more significant result and is more likely to be a true biological difference and thus validatable.

For a typical experiment with 3 biological replicate samples per group, look for p-value < 0.05 as significant result.

Sequence Verification

Always check to make sure the sequence of the QPCR assay you are using for validation is the exact same as the sequence we have listed on your data result from the microarray experiment.

miRBase[2] is the official database of miRNA sequence information on which almost all commercially available microarray content is based (including LC Sciences). This database is updated periodically to include newly discovered miRNAs but also to correct name and sequence errors. As a result, the same miRNA name may have a different sequence in different versions of the database. LC Sciences’ custom microarrays are updated in-sync with miRBase updates; however, QPCR assays routinely lag behind in updates to sequence information.

Additional, over time there have been changes to miRNA nomenclature rules. As an example, in 2012 miRBase phased out the miR/miR* nomenclature in favor of the -5p/-3p nomenclature. The human, mouse, and C. elegans miRNAs were updated in the miRBase version 18 release and the remaining species were changed with the version 19 release. (See the miRBase blog for more information on name changes to miRNA sequences.(http://www.mirbase.org/blog/)

Selection of Controls

Normalization to endogenous control genes is currently the most accurate method to correct for potential RNA input or reverse transcription (RT) efficiency biases. Careful selection of an appropriate set of controls is extremely important as significant variation has been observed between samples.

For each sample, we suggest using at least one, preferably two, small RNA controls from the following list.

Additionally, we recommend to add one or more miRNAs, which have similar expression levels in all your samples (based on the microarray data), to your control list. The following miRNAs have been found relatively stable in human tissues and NCI-60 cell lines. However, each experiment is different. For microarray expression data from LC Sciences, it is preferable that you select control miRNAs that have high signal intensities (>10,000).

Also, please refer to this Application Note from Applied Biosystems – “Endogenous Controls for Real-Time Quantitation of miRNA Using TaqMan^® MicroRNA Assays” for more on selection of controls for validation experiments.

A team of researchers from the University of Rochester and the University of Tainan, Taiwan used microarrays to screen miRNA profiles in response to vitamin D and found that a tumor suppressive miRNA, miR-98, is transcriptionally induced by 1α,25-dihydroxyvitamin D₃ (1,25-VD) in LNCaP. Mechanistic dissection revealed that 1,25-VD-induced miR-98 is mediated through both a direct mechanism, enhancing the VDR binding response element in the promoter region of miR-98, and an indirect mechanism, down-regulating LIN-28 expression. Knockdown of miR-98 led to a reduction of 1,25-VD anti-growth effect and overexpression of miR-98 suppressed the LNCaP cells growth via inducing G2/M arrest. And CCNJ, a protein controlling cell mitosis, is down-regulated by miR-98 via targeting 3′-untranslated region of CCNJ. Interestingly, miR-98 levels in blood are increased upon 1,25-VD treatment in mice suggesting the biomarker potential of miR-98 in predicting 1,25-VD response.

Biomarker potential of miRNAs relies on their high stability and existence in formalin-fixed tissue, cell free serum/plasma, and urine. The biological response to vitamin D varied among individuals. Factors contributing to a broad range of sensitivity to vitamin D include genomic polymorphisms of VDR and metabolism enzymes and pathological conditions altering VDR activity.

Biomarkers representing biological response of vitamin D treatment will be more meaningful than measurement of serum vitamin D level due to a broad range of sensitivity to vitamin D and other factors. Therefore the research team explored the biomarker potential of miR-98 representing vitamin D treatment in mice bearing prostate cancer. The cellular fraction of blood contains miR-98 whose expression correlated well with vitamin D treatment in both wild type and cancerous mice. Further investigation correlating expression levels of miR-98 and other vitamin D-regulated anti-tumor miRNAs (such as miR-22) in blood or other body fluid with anti-tumor effect of vitamin D is demanded.

Together, the finding that growth inhibitive miR-98 is induced by 1,25-VD provides a potential therapeutic target for prostate cancer and a potential biomarker for 1,25-VD anti-tumor action.

• Ting HJ, Messing J, Yasmin-Karim S, Lee YF. (2013) Identification of microRNA-98 as a therapeutic target inhibiting prostate cancer growth and a biomarker induced by vitamin D. J Biol Chem 288(1), 1-9. [abstract]

April 4th, 2013 – 11:00AM-1:30PM, Houston, TX

Title: Degradome Sequencing for Plant microRNA Target Identification

Date: Tuesday – 04/02/13

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post-transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules (targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA-target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.

Recently, degradome sequencing, a modified 5′-rapid amplification of cDNA ends (RACE) performed with next-gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation. This presentation will provide an overview of this emerging method for miRNA target identification, as well as practical information for performing degradome sequencing experiments and data analysis. Diverse case study examples will be provided to illustrate the usefulness of this latest application of next-gen sequencing technology to plant miRNA research.

Presented by
Dr. Qi Zhu

Senior Scientist

LC Sciences LLC

Webinar Outline

• This emerging method for miRNA target identification.
• Practical information for performing degradome sequencing experiments and data analysis.
• Diverse case study examples illustrating this latest application of next-gen sequencing technology to plant miRNA research.

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America – 1:00 Central Standard Time
https://attendee.gotowebinar.com/register/550047533582726400

Europe, Asia – 2:00 PM Greenwich Mean Time
https://attendee.gotowebinar.com/register/3791425404452347904

After registering, you will receive a confirmation email containing information about joining the webinar.

After completion of the webinar, you will receive an email containing a link to the archived webinar.

In a fascinating new study,  a team led by researchers at the University of Lethbridge, Canada show that the fetal transcriptome, through microRNA (miRNA) regulation, responds to prenatal stress in association with epigenetic signatures of psychiatric and neurological diseases. Pregnant Long-Evans rats were assigned to stress from gestational days 12 to 18 while others served as handled controls. Gestational stress in the dam disrupted parturient maternal behaviour and was accompanied by characteristic brain miRNA profiles in the mother and her offspring, and altered transcriptomic brain profiles in the offspring. In the offspring brains, prenatal stress upregulated miR-103, which is involved in brain pathologies, and downregulated its potential gene target Ptplb. Prenatal stress downregulated miR-145, a marker of multiple sclerosis in humans. Prenatal stress also upregulated miR-323 and miR-98, which may alter inflammatory responses in the brain. Furthermore, prenatal stress upregulated miR-219, which targets the gene Dazap1. Both miR-219 and Dazap1 are putative markers of schizophrenia and bipolar affective disorder in humans. Offspring transcriptomic changes included genes related to development, axonal guidance and neuropathology.

These findings indicate that prenatal stress modifies epigenetic signatures linked to disease during critical periods of fetal brain development. These observations provide a new mechanistic association between environmental and genetic risk factors in psychiatric and neurological disease.

• Zucchi FCR, Yao Y, Ward ID, Ilnytskyy Y, Olson DM, et al. (2013) Maternal Stress Induces Epigenetic Signatures of Psychiatric and Neurological Diseases in the Offspring. PLoS ONE 8(2),  e56967. [article]

Spodoptera frugiperda (army worm)

In the first study, identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using combination of deep sequencing and bioinformatics as well as experimental approaches¹. The predominant miRNAs were found conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori with 76 out of the 90 identified miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection and most of them were down-regulated after infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. The data presented here further support conservation of miRNAs in insects and other organisms and the results reveal differential expression of host miRNAs upon baculovirus infection suggesting their potential roles in host-virus interactions.

Plutella xylostella (diamondback moth larvae)

Next, small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum². Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.

1.  Mehrabadi M, Hussain M, Asgari S. (2013) MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection. J Gen Virol [Epub ahead of print]. [abstract]
2.  Etebari K, Hussain M, Asgari S. (2013) Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum. Insect Biochem Mol Biol [Epub ahead of print]. [abstract]

Hébert SS, Wang WX, Zhu Q, Nelson PT. (2013) A Study of Small RNAs from Cerebral Neocortex of Pathology-Verified Alzheimer’s Disease, Dementia with Lewy Bodies, Hippocampal Sclerosis, Frontotemporal Lobar Dementia, and Non-Demented Human Controls. J Alzheimers Dis [Epub ahead of print]. [abstract]

Mehrabadi M, Hussain M, Asgari S. (2013) MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection. J Gen Virol [Epub ahead of print]. [abstract]

microRNA Microarray

Han H, Peng J, Hong Y, Zhang M, Han Y, Liu D, Fu Z, Shi Y, Xu J, Tao J, Lin J. (2013) MicroRNA expression profile in different tissues of BALB/c mice in the early phase of Schistosoma japonicum infection. Mol Biochem Parasitol [Epub ahead of print]. [abstract ]

Zucchi FCR, Yao Y, Ward ID, Ilnytskyy Y, Olson DM, et al. (2013) Maternal Stress Induces Epigenetic Signatures of Psychiatric and Neurological Diseases in the Offspring. PLoS ONE 8(2),  e56967. [abstract]

Iwasaki YW, Kiga K, Kayo H, Fukuda-Yuzawa Y, Weise J, Inada T, Tomita M, Ishihama Y, Fukao T. (2013) Global microRNA elevation by inducible Exportin 5 regulates cell cycle entry. RNA [Epub ahead of print]. [abstract]

de la Morenaa MT,  Eitsonb JL, Dozmorovb IM, Belkayab S, Hooverb AR, Anguianoc E, Pascualc MV, van Oers NSC. (2013) Signature MicroRNA Expression Patterns Identified in Humans with 22q11.2 Deletion/DiGeorge Syndrome. Clin Immun [Epub ahead of print]. [abstract]

LC Sciences will be exhibiting at the upcoming Society of Toxicology – Annual Meeting in San Antonio, TX – March 10th-14th. Please visit us at booth #1064

The Society of Toxicology (SOT) 52nd Annual Meeting is the largest toxicology meeting and exhibition in the world, with an expected attendance of more than 7,300 scientists from academia, government, and industry from various countries around the globe. From the Plenary Opening Lecture and featured lectures to the wide range of scientific sessions and Continuing Education courses, the Annual Meeting offers an unparalleled depth of analysis and relevant toxicological issues. From basic to advanced topical issues, the thematic approach provides each attendee an opportunity to learn about emerging fields. Whether you are speaking in or chairing a session, honoring a colleague as the recipient of an SOT award, or collaborating with your peers at an SOT event, this meeting has something for every attendee. Plenary speaker includes Dr. Bruce Beutler (University of Texas Southwestern Medical Center, Dallas, Texas) and Prof. Jeremy K. Nicholson, Imperial College London, United Kingdom.

Presenter Name: Dr. Qi Zhu

Presentation Title: Degradome Sequencing for Plant microRNA Target Identification.

Presentation Abstract:

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post-transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules (targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA-target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.

Recently, degradome sequencing, a modified 5′-rapid amplification of cDNA ends (RACE) performed with next-gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation. This presentation will provide an overview of this emerging method for miRNA target identification, as well as practical information for performing degradome sequencing experiments and data analysis. Diverse case study examples will be provided to illustrate the usefulness of this latest application of next-gen sequencing technology to plant miRNA research.

In one study, researchers employed high-throughput sequencing and degradome analysis to identify miRNAs and their targets using high-throughput sequencing in cytoplasmic male sterility (CMS) and its maintainer fertile (MF) lines of Brassica juncea¹.

The researchers identified 197 known and 78 new candidate miRNAs during reproductive development of B. juncea. A total of 47 differentially expressed miRNAs between CMS and its MF lines were discovered, according to their sequencing reads number. Targeted genes of the miRNAs were identified by high-throughput sequencing and degradome approaches, including auxin response factor, and a host of transcription factors, which were observed to be differentially expressed between CMS and MF.

These findings we suggested miRNA might participate in the regulatory network of CMS by tuning fork in gene expressions in CMS B. juncea. The differential expression of miRNAs observed between CMS and MF lines suggested that biogenesis of miRNAs could be influenced in the CMS.

In another study, researchers sequenced three small RNA populations and a degradome of wild rice (O. rufipogon) by the Illumina sequencing platform and investigated the expression levels of microRNAs (miRNAs) by miRNA microarray². A de novo O. rufipogon genome was assembled using 55× coverage of raw sequencing data and a total of 387 miRNAs were identified in the O. rufipogon. Of these, O. rufipogon miRNAs, 259 were not found in cultivated rice, suggesting a loss of these miRNAs in the cultivated rice. The team also found that 48 miRNAs were novel in the cultivated rice, suggesting that they were potential targets of domestication selection.

Some miRNAs showed significant expression differences between wild and cultivated rice, suggesting that expression of miRNA could also be a target of domestication. Their results illustrated that miRNA genes, like protein-coding genes, might have been significantly shaped during rice domestication and could be one of the driving forces that contributed to rice domestication.

1. Yang J, Liu X, Xu B, Zhao N, Yang X, Zhang M. (2013) Identification of miRNAs and their targets using high-throughput sequencing and degradome analysis in cytoplasmic male-sterile and its maintainer fertile lines of brassica juncea. BMC Genomics 14(1), 9. [article]
2. Wang Y, Bai X, Yan C, Gui Y, Wei X, Zhu QH, Guo L, Fan L. (2012) Genomic dissection of small RNAs in wild rice (Oryza rufipogon): lessons for rice domestication. New Phytol 196(3), 914-25. [abstract]

microRNA: Targets and Tools for Therapeutic Development

microRNAs are playing an important role in a range of diseases such as cancer, metabolic diseases and cardiovascular disorders. Their involvement in many physiological pathways, makes them an excellent target for pharmaceutical drug development and their use as biomarkers or for profiling and screening turns them into a valuable tool for diagnostics. This conference highlights the latest developments in using miRNA’s for health and disease, the novel applications and progress made in manipulating microRNA, and the opportunities for using microRNA as a tool.

20+ SCIENTIFIC PRESENTATIONS COVERING:

• Therapeutic Potential in Inflammation, Cancer and Pain
• New Technological Strategies
• microRNAs as Biomarkers and in Diagnostics
• Tissue-Based Analysis
• Circulating miRNAs

(find out more…)

Epitope Mapping on a Flexible Microfluidic Chip Platform

Understanding antibody specificity at the molecular level provides the key to optimizing their use as research or diagnostic tools – it forms the basis of their application as therapeutic agents. Arrays of protein sequence-derived short peptides have emerged as a powerful tool to identify and characterize such binding epitopes.

• Microfluidic platform technology overview
• Experimental design and options
• Case studies and application examples

This webinar was recorded 1/12/13.

Download a copy of the presentation slides

Feb 1st, 2013 – 10:00AM – 2:30 PM
Marriott Medical Center Ballroom,
Texas Medical Center, Houston TX

Visit LC Sciences at Table #16

See new lab products, analytical equipment, and learn about new research techniques. The Houston BioResearch Product Faire™ boasts a wide range of relevant and high quality vendors to ensure that you find the products you need to meet your research needs.

(read more…)

Etebari K, Hussain M, Asgari S. (2013) Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum. Insect Biochem Mol Biol [Epub ahead of print]. [abstract]

Degradome Sequencing

Yang J, Liu X, Xu B, Zhao N, Yang X, Zhang M. (2013) Identification of miRNAs and their targets using high-throughput sequencing and degradome analysis in cytoplasmic male-sterile and its maintainer fertile lines of brassica juncea. BMC Genomics 14(1), 9. [abstract]

microRNA Microarray

Wang Y, Vogel G, Yu Z, Richard S. (2013) The QKI-5 and QKI-6 RNA binding proteins regulate the expression of miR-7 in glial cells. Mol Cell Biol [Epub ahead of print]. [abstract]

Title: Epitope Mapping on a Flexible Microfluidic Chip Platform

Date: Tuesday –  01/29/13

Understanding antibody specificity at the molecular level provides the key to optimizing their use as research or diagnostic tools – it forms the basis of their application as therapeutic agents. Arrays of protein sequence-derived short peptides have emerged as a powerful tool to identify and characterize such binding epitopes. LC Sciences combines the flexibility of customizable parallel synthesis and high data quality of microfluidic technologies to create high density peptide microarrays for high throughput detection, concentration titration, and screening applications.

Presented by
Dr. Christoph Eicken

Head of Technical Services

LC Sciences LLC

Webinar Outline

• Microfluidic platform technology overview
• Experimental design and options
• Case studies and application examples

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America  – 1:00 Central Standard Time
https://attendee.gotowebinar.com/register/6577930914452847872                                            

Europe, Asia – 2:00 PM Greenwich Mean Time
https://attendee.gotowebinar.com/register/2491584954707092480

After registering, you will receive a confirmation email containing information about joining the webinar.

After completion of the webinar, you will receive an email containing a link to the archived webinar.

Garcinol synergizes with gemcitabine to inhibit cell proliferation and induce apoptosis in PaCa cells with significant modulation of key cancer regulators including PARP, VEGF, MMPs, ILs, caspases, and NF-κB. In addition, biostatistical analyses, quantitative reverse transcription PCR data, and in silico modeling using TargetScan5, PicTar, and DNA intelligent analysis, microT-V.B4 database showed that these two agents modulated a number of microRNAs (miR-21, miR-196a, miR-495, miR-605, miR-638, and miR-453) linked to various canonical oncogenic signaling pathways.

MicroRNA microarrays showed that garcinol and/or gemcitabine treatment alters miRNA profile in PaCa cells. Heat maps (Fig. 3A, B, and C) generated by miRNA microarray analysis revealed expression of several miRNAs that are dysregulated in Panc-1 upon treatment. Figure 3D shows a volcano scatter plot with relative expression of miRNAs in all three treatment groups normalized to that of untreated control cells. A threshold of p < 0.05 (dotted line) was set as a selection criterion. Since garcinol showed a more potent effect in combination with gemcitabine in Panc-1 cells, this cell line was used as a model for further evaluation.

The authors have identified garcinol-specific miRNA biomarkers that sensitize PaCa cells to gemcitabine treatment, thus attenuating the drug-resistance phenotype. These results prompt further interest in garcinol and gemcitabine combination strategy as a drug modality to improve treatment outcome in patients diagnosed with PaCa.

• Parasramka MA, Ali S, Banerjee S, Deryavoush T, Sarkar FH, Gupta S. (2013) Garcinol sensitizes human pancreatic adenocarcinoma cells to gemcitabine in association with microRNA signatures. Mol Nutr Food Res [Epub ahead of print]. [abstract]

In the next study, a team led by researchers at Russian Academy of Medical Sciences used microarrays to analyze amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in Human cerebrospinal fluid (CSF) and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer’s disease (AD) and age-matched control neocortex². CSF, produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease.

1. Parsons XH, Parsons JF, Moore DA (2012) Genome-Scale Mapping of MicroRNA Signatures in Human Embryonic Stem Cell Neurogenesis. Mol Med Ther [Epub ahead of print]. [article]
2. Alexandrov PN, Dua P, Hill JM, Bhattacharjee S, Zhao Y, Lukiw WJ. (2012) microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF). Int J Biochem Mol Biol 3(4), 365-373. [article]

Poster Title: Degradome Sequencing for Plant miRNA Target Identification.

Presenter Name: Dr. Qi Zhu

Poster Abstract:

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post-transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules (targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA-target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.

In plants, miRNAs exert negative regulatory control by binding to and causing cleavage of their targets at the position between nucleotides 10 and 11 of the miRNA. Recently, degradome sequencing, a modified 5′-rapid amplification of cDNA ends (RACE) performed with next-gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation.  Deep sequencing of the 5’ ends of RNA degradation products allows identification of over-represented 5’ ends (miRNA cleavage sites) within and RNA sample. Matching cleavage sites to known miRNA sequences links miRNAs to their targets.

This poster will provide an overview of this emerging method for miRNA target identification, as well as case study examples to illustrate the usefulness of this latest application of next-gen sequencing technology to plant miRNA research.

Download the full size poster at: http://www.lcsciences.com/documents/degradome-sequencing-poster.pdf

LC Sciences will be exhibiting at the upcoming Plant & Animal Genomes Conference, Jan 12th-16th 2013 in San Diego, California.

The Largest Ag-Genomics Meeting in the World

PAGXXI brings together over 2,800 leading genetic scientists and reseachers in plant and animal research, and over 120 exhibits, 140+ workshops, 1000+ posters and 1700+ abstracts.

Visit us at booth #509 to hear about some of the things we have been doing with microRNA sequencing and degradome sequencing for our customers in the agricultural sciences.  We hope to see you there!

Degradome Sequencing Workshop

LC Sciences will be presenting a workshop along with Norgen Biotek covering Plant RNA extraction and Degradome Sequencing

This workshop will provide an overview of the degradome sequencing method for miRNA target identification, as well as practical information for performing degradome sequencing experiments.  Additionally, we will discuss the importance of miRNA sample preparation from various plant species/tissue types for downstream applications including next-gen sequencing, microarray and others. Diverse case study and application examples will be provided.

The research team identified 637 unique miRNA sequences in a large-scale screen for miRNA expression profiles in the developing lower deciduous molars of miniature pigs (Sus scrofa) using Illumina Solexa deep sequencing. Deep sequencing yields results that broadly cover genome-wide miRNAs from samples of various temporal and special origins without the need for any prior sequence knowledge. However, due to the relatively high cost and low throughput nature of sample handling, the systematic follow through of the sequencing discoveries for validation and/or profiling in a reproducible manner could take a long time and be expensive by repetitive sequencing.

Instead, a microarray can be used for high-throughput expression analysis. Microarrays are effective tools to capture the comprehensive sequencing information and to systematically profile and compare the gene expression based on the sequencing findings rapidly, reproducibly, and cost effectively.

The candidate miRNAs, discovered through sequencing, along with 105 known Sus scrofa miRNAs were included in a custom-designed miRNA microarray and used to analyze the miRNA expression profile in the bud, cap, early bell, and late bell stages of tooth development. Microarray analysis revealed 166 transcripts that were differentially expressed in the four stages. Bioinformatic analysis identified 18 key miRNAs, including let-7f, miR-128, miR-200b, and miR-200c, that might play key roles in tooth development.

LC Sciences is a leader in miRNA microarray profiling offering flexible services and delivering high quality results based on our innovative µParaflo® custom microarray platform. We have developed complementary bioinformatics tools necessary for extracting miRNA and miRNAome information from large sequencing data sets and for designing custom arrays.

• Li A, Song T, Wang F, Liu D, Fan Z, Zhang C, He J, Wang S. (2012) MicroRNAome and Expression Profile of Developing Tooth Germ in Miniature Pigs. PLoS One 7(12):e52256. [article]

Bioinformatic and subsequent small RNA northern blot analysis identified eight miRNA families (five conserved and three newly identified) differentially expressed under the N-deficient condition. Predicted and degradome-validated targets of the newly identified miRNAs suggest their involvement in a broad range of cellular responses and metabolic processes.

• Zhao M, Tai H, Sun S, Zhang F, Xu Y, Li WX. (2012) Cloning and characterization of maize miRNAs involved in responses to nitrogen deficiency. PLoS One 7(1), e29669. [article]

In plants, miRNAs not only post-transcriptionally regulate their own targets but also interact with each other in regulatory networks to affect many aspects of development, such as growth, development and responses to biotic and abiotic stresses. Hundreds of miRNAs have been identified in higher plants by direct cloning or more recently by next-gen sequencing. To determine the function of these miRNAs we must first identify their targets.

Originally, plant miRNA targets have been studied via computational prediction, which is based on either perfect or near-perfect sequence complementarity between miRNA and the target mRNA or sequence conservation among different species. However, target prediction is very challenging, especially when a high level of mismatches exists in miRNA:target pairing.

Recently, a new method called degradome sequencing, which combines high-throughput RNA sequencing with bioinformatic tools, has-been successfully established to screen for miRNA targets in plants. Using degradome sequencing, many of the previously validated and predicted targets of miRNAs have been verified indicating that it is an efficient strategy to identify smRNA targets on a large scale in plants.

Degradome sequencing reveals miRNA targets by globally identifying the remnants of small RNA-directed target cleavage by sequencing the 5′ ends of uncapped RNAs. Sequencing reads are mapped to mRNAs and the 5′ terminal nucleotide of miRNA-cleaved mRNA fragments corresponds to the nucleotide that is complementary to the 10th nucleotide of the miRNA. Therefore, the cleaved RNA targets have distinct peaks in the degradome sequence reads at the predicted cleavage site relative to other regions of the transcript. Confirmed miRNA targets are presented in the form of target plots (t-plots).

Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases. However, unlike Arabidopsis and other plants, much less is known about its miRNAs and their targets. Now, researchers at the Chinese Academy of Agricultural Sciences, Beijing have used Illumina high-throughput sequencing analysis of small RNAs as well as degradome sequencing of B. napus to identify novel miRNAs and miRNA targets¹.

Forty-one conserved B. napus miRNAs and 62candidate novel B. napus-specific miRNAs were identified through small RNA sequencing and further verified by real-time RT-PCR. A total of 33 non-redundant target ESTs for 25conserved miRNAs, and 19 non-redundant target ESTs for 17 B. napus-specific miRNAs were identified through degradome sequencing and verified by RNA ligase-mediated 5’RACE mapping. This study describes large scale sequencing and identification of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.

miRNAs also play important regulatory roles in development and stress response in plants. Wild soybean (Glycine soja) has undergone long-term natural selection and may have evolved special mechanisms to survive stress conditions as a result. However, little information about miRNAs especially miRNAs responsive to aluminum (Al) stress is available in wild soybean.

Now, researchers at the South China Agricultural University, Guangzhou have sequenced two small RNA libraries and two degradome libraries constructed from the roots of Al-treated and Al-free G. soja seedlings². Among all the identified miRNAs, the expressions of 30 miRNAs were responsive to Al stress. Through degradome sequencing, 86 genes were identified as targets of the known miRNAs and five genes were found to be the targets of the novel miRNAs obtained in this study. Gene ontology (GO) annotations of target transcripts indicated that 52 target genes cleaved by conserved miRNA families might play roles in the regulation of transcription. Additionally, some genes known to be responsive to stress, were found to be cleaved under Al stress conditions. These findings provide valuable information to understand the function of miRNAs in Al tolerance.

Cucumber (Cucumis sativus) is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, a team led by researchers at Zhejiang University, Hangzhou constructed two independent small RNA libraries from cucumber leaves and roots, respectively, and sequenced them with the high-throughput Illumina system³. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified.

With the recently developed ‘high throughput degradome sequencing’ approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. This study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

1. Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ, Sun J, Fan YL, Wang L. (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421. [article]
2. Qiao-Ying Z, Cun-Yi Y, Qi-Bin M, Xiu-Ping L, Wen-Wen D, Hai N. (2012) Identification of wild soybean miRNAs and their target genes responsive to aluminum stress. BMC Plant Biol 12(1), 182. [article]
3. Mao W, Li Z, Xia X, Li Y, Yu J. (2012) A Combined Approach of High-Throughput Sequencing and Degradome Analysis Reveals Tissue Specific Expression of MicroRNAs and Their Targets in Cucumber. PLoS One 7(3), e33040. [article]

LC Sciences offers comprehensive services for both small RNA sequencing and degradome sequencing using the Illumina next-gen sequencing platform and in-house developed bioinformatics.

LC Sciences will be exhibiting at the upcoming Plant & Animal Genomes Conference, Jan 12th-16th 2013 in San Diego, California.

The Largest Ag-Genomics Meeting in the World

PAGXXI brings together over 2,800 leading genetic scientists and reseachers in plant and animal research, and over 120 exhibits, 140+ workshops, 1000+ posters and 1700+ abstracts.

Visit us at booth #509 to hear about some of the things we have been doing with microRNA sequencing and degradome sequencing for our customers in the agricultural sciences.  We hope to see you there!

Visit us at booth #1412 and learn how the study of microRNA is advancing the field of Cell Biology.

Recent Customer Cell Biology Publications

Tumaneng K, Schlegelmilch K, Russell RC, Yimlamai D, Basnet H, Mahadevan N, Fitamant J, Bardeesy N, Camargo FD, Guan KL. (2012) YAP mediates crosstalk between the Hippo and PI(3)K-TOR pathways by suppressing PTEN via miR-29. Nat Cell Biol [Epub ahead of print]. [abstract]

Hu S, Wilson KD, Ghosh Z, Han L, Wang Y, Lan F, Ransohoff KJ, Wu JC. (2012) MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2. Stem Cells [Epub ahead of print]. [article]

Pencheva N, Tran H, Buss C, Huh D, Drobnjak M, Busam K, Tavazoie SF. (2012) Convergent Multi-miRNA Targeting of ApoE Drives LRP1/LRP8-Dependent Melanoma Metastasis and Angiogenesis. Cell [Epub ahead of print]. [abstract]

Liu J, Githinji J, McLaughlin B, Wilczek K, Nolta J.(2012) Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell-Derived Neural Stem Cells. Stem Cell Rev [Epub ahead of print]. [abstract]

Afonyushkin T,Oskolkova OV,Bochkov VN. (2012) Permissive role of miR-663 in induction of VEGF and activation of the ATF4 branch of unfolded protein response in endothelial cells by oxidized phospholipids. Atherosclerosis [Epub ahead of print]. [article]

Read D, Deepti A, Cawley K, Gupta A, Oommen D, Verfaillie T, Matus S, Agostinis P, Hetz C, Samali A. (2012) Perk-dependent Repression of miR-106b-25 Cluster is Required for ER stress-induced Apoptosis. Cell Death Dis [Epub ahead of print]. [article]

Researchers at the Xiangya Hospital of Central South University, China have used a microRNA microarray to show in vitro, that miR-148a is dramatically upregulated during osteoclastic differentiation of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-148a in CD14+ PBMCs promoted osteoclastogenesis, while inhibition of miR-148a attenuated osteoclastogenesis. MiR-148a directly targeted MAFB mRNA, a transcription factor negatively regulating osteoclastogenesis, by binding to the 3′UTR and repressed MAFB protein expression.

Additionally, in vivo, their study showed that silencing of miR-148a using a specific antagomir inhibited bone resorption and increased bone mass in both ovariectomy (OVX) and sham-operated control mice.

Finally, in patients, the results showed that miR-148a levels significantly increased in CD14+ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower BMD in lupus patients compared with normal control. Thus, our study provided a new insight into the roles of miRNAs in osteoclastogenesis, and contributed to a new therapeutic way for osteoporosis.

• Cheng P, Chen C, He HB, Hu R, Zhou HD, Xie H, Zhu W, Dai RC, Wu XP, Liao EY, Luo XH. (2012) MiR-148a regulates osteoclastogenesis via targeting MAFB. J Bone Miner Res [Epub ahead of print]. [abstract]

MicroRNA directs bone morphogenetic cell fate decision

A team led by researchers at University of Genova, Italy have shown that silencing of the regulatory protein KSRP in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. Gene expression was analyzed by transcriptome sequencing (RNA-seq).

Additonally, they made the striking observation that miR-206 re-expression in KSRP-silenced cells is sufficient to enhance myogenin expression and to abrogate the osteoblastic phenotype.

They further demonstrate that SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage.

• Pasero M, Giovarelli M, Bucci G, Gherzi R, Briata P. (2012) Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation. Cell Rep 2(5), 1159-68. [article]

MicroRNA’s role in gamma-irradiation osteoblast cell death

To further understand the mechanisms of radiation-induced damage in different cells, researchers at the Uniformed Services University of the Health Sciences performed microRNA (miRNA)-arrays using purified miRNAs from human fetal osteoblastic (hFOB) cells before and post-irradiation.

They found that radiation inhibited miR-30c expression (which has potential target sites on the REDD1 gene) in hFOB cells so they explored the effects of miR-30c on REDD1 expression using miR-30c inhibitor and precursor (pre-miR-30c). The results show that pre-miR-30c transfection suppressed REDD1 expression in 14 day cultured hFOB cells and resulted in hFOB cell death.

Their data suggest that miR-30c plays a key role in radiation-induced cell damage which might be through regulation of REDD1 expression.

• Li XH, Ha CT, Fu D, Xiao M (2012) Micro-RNA30c Negatively Regulates REDD1 Expression in Human Hematopoietic and Osteoblast Cells after Gamma-Irradiation. PLoS ONE 7(11), e48700. [article]

Analysis of the miRNA expression profile in tissue specimens from gastric MALT lymphomas and surrounding non-tumor mucosae revealed that a hematopoietic-specific miRNA miR-142 and an oncogenic miRNA miR-155 were overexpressed in MALT lymphoma lesions. The expression levels of miR-142-5p and miR-155 were significantly increased in MALT lymphomas which do not respond to Helicobacter pylori (H. pylori) eradication. The expression levels of miR-142-5p and miR-155 were associated with the clinical courses of gastric MALT lymphoma cases.

The results of this study indicate that overexpression of miR-142-5p and miR-155 plays a critical role in the pathogenesis of gastric MALT lymphoma. These miRNAs might have potential application as therapeutic targets and novel biomarkers for gastric MALT lymphoma.

A team of researchers at the China Agricultural University, Beijing, and Iowa State University used next-gen sequencing performed by LC Sciences to investigate Marek’s disease virus (MDV)-induced T-cell lymphoma in chicken, which poses a serious threat to poultry health².

Analysis of host miRNA expression profiles in two MDV-infected samples (tumorous spleen and MD lymphoma from liver), and two non-infected controls (non-infected spleen and lymphocytes) identified 187 and 16 known miRNAs in chicken and MDV, respectively. They identified 28 down-regulated miRNAs and 11 up-regulated miRNAs in MDV-infected samples by bioinformatic analysis. The gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-199*, and gga-miR-140* were down-regulated, and gga-miR-146c was up-regulated in MDV-infected tumorous spleens and MD lymphomas.

Differential expression of these miRNAs and their predicted targets strongly suggest that they contribute to MDV-induced lymphomagenesis.

1. Saito Y, Suzuki H, Tsugawa H, Imaeda H, Matsuzaki J, et al. (2012) Overexpression of miR-142-5p and miR-155 in Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Resistant to Helicobacter pylori Eradication. PLoS ONE 7(11): e47396. [article]
2. Lian L, Qu L, Chen Y, Lamont SJ, Yang N (2012) A Systematic Analysis of miRNA Transcriptome in Marek’s Disease Virus-Induced Lymphoma Reveals Novel and Differentially Expressed miRNAs. PLoS ONE 7(11): e51003. [article]

Download the presentation slides.

microRNAs ‐ What We Know

1. All miRNAs are small non‐coding RNAs, usually consisting of…

Now, a team of researchers at Tulane and Xaiver Universities in New Orleans have defined, for the first time, specific miRNAs under the direct regulation of IGF-1 signaling in the estrogen receptor positive MCF-7 breast cancer cell line and demonstrate kinase signaling as a modulator of expression for a small subset of microRNAs.

Through the use of miRNA microarray profiling, they demonstrate a subset of previously described tumor suppressor and oncogenic microRNAs (miRNAs) that are under the direct regulation of IGF-1 signaling. Additionally, they show that the selective inhibition of either the MAPK or AKT pathways prior to IGF-1 stimulation prevents the expression of previously described tumor suppressor miRNAs that are family and cluster specific.

Martin EC, Bratton MR, Zhu Y, Rhodes LV, Tilghman SL, et al. (2012) Insulin-Like Growth Factor-1 Signaling Regulates miRNA Expression in MCF-7 Breast Cancer Cell Line. PLoS ONE 7(11): e49067. [article]

Workshop Summary:

This workshop will provide an overview of the degradome sequencing method for miRNA target identification, as well as practical information for performing degradome sequencing experiments.  Additionally, we will discuss the importance of miRNA sample preparation from various plant species/tissue types for downstream applications including next-gen sequencing, microarray and others. Diverse case study and application examples will be provided.

Presenter : Dr. Qi Zhu

Presentation Title: Degradome Sequencing for Plant microRNA Target Identification.

Presentation Abstract:

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post-transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules (targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA-target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.

Recently, degradome sequencing, a modified 5′-rapid amplification of cDNA ends (RACE) performed with next-gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation. This presentation will provide an overview of this emerging method for miRNA target identification, as well as practical information for performing degradome sequencing experiments and data analysis. Diverse case study examples will be provided to illustrate the usefulness of this latest application of next-gen sequencing technology to plant miRNA research.

Presenters: Dr. Won-Sik Kim & Nezar Rghei

Presentation Title: The Importance Of Sample Preparation For Down Stream Applications: NGS, Sequencing, Microarray, RT-PCR, MdX, etc.

Presentation Abstract:

MicroRNAs are endogenous 20 to 24 nucleotide noncoding RNAs that play crucial posttranscriptional regulatory roles in plant and animals. Tremendous efforts are currently being undertaken to understand the profile of the entire miRNA population of a biological sample, which will provide useful information on miRNA activity. Many miRNA discovery tools, including micro arrays and Next-gen-based sequencing, have made it possible to comprehensively and accurately assess the entire miRNA repertoire. This presentation deals with the importance of sample preparation on downstream applications. A prerequisite for obtaining successful results from these approaches is an efficient method for total RNA purification without bias. The choice of the method of RNA purification is critical to the outcome of downstream analysis. This is made more significant in variations of the plant specimens and the high phenolics, starch and other inhibitors co-isolating with the RNA. The three most popular RNA purification methods (spin columns using Silicon Carbide, spin columns employing silica membrane and phenol/chloroform extraction) are compared in this talk in terms of quality, quantity and small RNA recovery from difficult and moderately challenging plant samples. Examples of microRNA study cases will be also discussed to highlight the importance of the RNA purification method used for different plant species.

Our customers, through a combination of next-gen sequencing and microarray experiments have take taken large steps towards this end and provided significant contributions to the miRNA body of knowledge for this important animal.

Guo XK, Zhang Q, Gao L, Li N, Chen XX, Feng WH. (2012) Additional expression of microRNA-181 inhibits Porcine Reproductive and Respiratory Syndrome Virus replication and its implications for controlling virus infection. J Virol [Epub ahead of print]. [abstract]

Li R, Sun Q, Jia Y, Cong R, Ni Y, et al. (2012) Coordinated miRNA/mRNA Expression Profiles for Understanding Breed-Specific Metabolic Characters of Liver between Erhualian and Large White Pigs. PLoS ONE 7(6), e38716. [abstract]

Lin F, Li R, Pan Zx, Zhou B, Yu Db, et al. (2012) miR-26b Promotes Granulosa Cell Apoptosis by Targeting ATM during Follicular Atresia in Porcine Ovary. PLoS ONE 7(6), e38640. [abstract]

Li M, Liu Y, Wang T, Guan J, Luo Z, Chen H, Wang X, Chen L, Ma J, Mu Z, Jiang AA, Zhu L, Lang Q, Zhou X, Wang J, Zeng W, Li N, Li K, Gao X, Li X (2011) Repertoire of Porcine MicroRNAs in Adult Ovary and Testis by Deep Sequencing. Int J Biol Sci 7(7), 1045-55. [abstract]

Li M, Xia Y, Gu Y, Zhang K, Lang Q, et al. (2010) MicroRNAome of Porcine Pre- and Postnatal Development. PLoS ONE 5(7), e11541. [abstract

Zhou B, Liu HL, Shi FX, Wang JY. (2010) MicroRNA expression profiles of porcine skeletal muscle. Anim Genet 41(5), 499-508. [abstract]

Luo L, Ye L, Liu G, Shao G, Zheng R, Ren Z, Zuo B, Xu D, Lei M, Jiang S, Deng C, Xiong Y, Li F. (2010) Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes. PLoS ONE 5(8), e11744. [abstract]

Curry E, Ellis SE, Pratt SL. (2009) Detection of porcine sperm microRNAs using a heterologous microRNA microarray and reverse transcriptase polymerase chain reaction. Mol Reprod Dev 76 (3), 218-19. [abstract]

LC Sciences will be exhibiting at the upcoming Plant & Animal Genomes Conference, Jan 12th-16th 2013 in San Diego, California.

The Largest Ag-Genomics Meeting in the World

PAGXXI brings together over 2,800 leading genetic scientists and reseachers in plant and animal research, and over 120 exhibits, 140+ workshops, 1000+ posters and 1700+ abstracts.

Visit us at booth #509 to hear about some of the things we have been doing with microRNA sequencing and degradome sequencing for our customers in the agricultural sciences.  We hope to see you there!

• Pasero M, Giovarelli M, Bucci G, Gherzisend R, Briatasend P. (2012) Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation. Cell Reports [Epub ahead of print]. [abstract]

Scientists at the University of Pittsburgh School of Medicine demonstrate a technical advance that provides the means for generating a renewable resource of pure human cardiovascular cells that can be used to dissect the mechanism of human inherited heart disease, and for the future development of drug and cell therapeutics for heart disease.

• Lin B, Kim J, Li Y, Pan H, Carvajal-Vergara X, Salama G, Cheng T, Li Y, Lo CW, Yang L. (2012) High-purity enrichment of functional cardiovascular cells from human iPS cells. Cardiovasc Res [Epub ahead of print]. [abstract]

In plants, miRNAs not only post-transcriptionally regulate their own targets but also interact with each other in regulatory networks to affect many aspects of development, such as growth, development and responses to biotic and abiotic stresses. Hundreds of miRNAs have been identified in higher plants by direct cloning or more recently by next-gen sequencing. To determine the function of these miRNAs we must first identify their targets.

Originally, plant miRNA targets have been studied via computational prediction, which is based on either perfect or near-perfect sequence complementarity between miRNA and the target mRNA or sequence conservation among different species. However, target prediction is very challenging, especially when a high level of mismatches exists in miRNA:target pairing.

Recently, a new method called degradome sequencing, which combines high-throughput RNA sequencing with bioinformatic tools, has-been successfully established to screen for miRNA targets in plants. Using degradome sequencing, many of the previously validated and predicted targets of miRNAs have been verified indicating that it is an efficient strategy to identify smRNA targets on a large scale in plants.

Degradome sequencing reveals miRNA targets by globally identifying the remnants of small RNA-directed target cleavage by sequencing the 5′ ends of uncapped RNAs. Sequencing reads are mapped to mRNAs and the 5′ terminal nucleotide of miRNA-cleaved mRNA fragments corresponds to the nucleotide that is complementary to the 10th nucleotide of the miRNA. Therefore, the cleaved RNA targets have distinct peaks in the degradome sequence reads at the predicted cleavage site relative to other regions of the transcript. Confirmed miRNA targets are presented in the form of target plots (t-plots).

Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases. However, unlike Arabidopsis and other plants, much less is known about its miRNAs and their targets. Now, researchers at the Chinese Academy of Agricultural Sciences, Beijing have used Illumina high-throughput sequencing analysis of small RNAs as well as degradome sequencing of B. napus to identify novel miRNAs and miRNA targets¹.

Forty-one conserved B. napus miRNAs and 62candidate novel B. napus-specific miRNAs were identified through small RNA sequencing and further verified by real-time RT-PCR. A total of 33 non-redundant target ESTs for 25conserved miRNAs, and 19 non-redundant target ESTs for 17 B. napus-specific miRNAs were identified through degradome sequencing and verified by RNA ligase-mediated 5’RACE mapping. This study describes large scale sequencing and identification of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.

miRNAs also play important regulatory roles in development and stress response in plants. Wild soybean (Glycine soja) has undergone long-term natural selection and may have evolved special mechanisms to survive stress conditions as a result. However, little information about miRNAs especially miRNAs responsive to aluminum (Al) stress is available in wild soybean.

Now, researchers at the South China Agricultural University, Guangzhou have sequenced two small RNA libraries and two degradome libraries constructed from the roots of Al-treated and Al-free G. soja seedlings². Among all the identified miRNAs, the expressions of 30 miRNAs were responsive to Al stress. Through degradome sequencing, 86 genes were identified as targets of the known miRNAs and five genes were found to be the targets of the novel miRNAs obtained in this study. Gene ontology (GO) annotations of target transcripts indicated that 52 target genes cleaved by conserved miRNA families might play roles in the regulation of transcription. Additionally, some genes known to be responsive to stress, were found to be cleaved under Al stress conditions. These findings provide valuable information to understand the function of miRNAs in Al tolerance.

Cucumber (Cucumis sativus) is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, a team led by researchers at Zhejiang University, Hangzhou constructed two independent small RNA libraries from cucumber leaves and roots, respectively, and sequenced them with the high-throughput Illumina system³. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified.

With the recently developed ‘high throughput degradome sequencing’ approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. This study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

1. Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ, Sun J, Fan YL, Wang L. (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421. [article]
2. Qiao-Ying Z, Cun-Yi Y, Qi-Bin M, Xiu-Ping L, Wen-Wen D, Hai N. (2012) Identification of wild soybean miRNAs and their target genes responsive to aluminum stress. BMC Plant Biol 12(1), 182. [article]
3. Mao W, Li Z, Xia X, Li Y, Yu J. (2012) A Combined Approach of High-Throughput Sequencing and Degradome Analysis Reveals Tissue Specific Expression of MicroRNAs and Their Targets in Cucumber. PLoS One 7(3), e33040. [article]

LC Sciences offers comprehensive services for both small RNA sequencing and degradome sequencing using the Illumina next-gen sequencing platform and in-house developed bioinformatics.

We apologize for the inconvenience but due to illness, we have had to postpone our upcoming cardiovascular microRNA webinar.

The webinar will be delayed exactly one week.  The new date will be: Tuesday – 12/04/12

If you have already registered for the webinar, there is nothing you need to do.  You will remain registered for the webinar on the new date.  If you have not yet registered, you can register here for one of the webinars on the new date.

Title: Recent Work & Current Methods in Cardiovascular microRNA Research

Date: Tuesday – 12/04/12

Presented by
Dr. Christoph Eicken

Head of Technical Services

LC Sciences LLC

MicroRNAs (miRNAs) are non-coding small RNAs (18–25 nt) that play crucial roles in various biological processes, including development, as gene regulators. In this study, researchers at the University of Queensland, Australia explored the possibility of utilizing synthetic inhibitor and mimic of a miRNA by oral feeding and confirmed that the inhibitor and mimic can successfully inhibit/oversupply har-miR-2002b in Helicoverpa armigera (cotton bollworm) larvae.

Bioassays by oral feeding indicated 70% reduction in fecundity and 40% larval mortality in the presence of har-miR-2002b mimic. In addition, a large proportion of those larvae that made it to the pupal stage were deformed and only few moths emerged from them. This implied an important role of har-miR-2002b in larval development and adult fecundity.

• Jayachandran B, Hussain M, Asgari S. (2012) An insect trypsin-like serine protease as a target of miRNA: utilization of miRNA mimics and inhibitors by oral feeding. Insect Biochem Mol Biol [Epub ahead of print]. [abstract]

Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear.

In the latest issue of Nature Cell Biology, they provide evidence that YAP activates the mammalian target of rapamycin (mTOR), identify the tumour suppressor PTEN as a critical mediator of YAP in mTOR regulation, demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation, and finally show that PI(3)K-mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia.

Researchers at Stanford University demonstrate a feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4 that represents an important new mechanism for understanding and inducing pluripotency in somatic cells².

They used global bioinformatics analysis of miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Their data, recently published in Stem Cells, show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of the miR-302,

They obtained higher reprogramming efficiencies of human adipose-derived stem cells (hASCs) into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as “KMOS3″) when compared to using four factors (“KMOS”). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency.

Researchers at Rockefeller University have uncovered a convergent and cooperative miRNA network that drives melanoma metastasis³.

In the latest issue of Cell, they identify miR-1908, miR-199a-5p, and miR-199a-3p as endogenous promoters of metastatic invasion, angiogenesis, and colonization in melanoma.

They further  identify miRNAs with dual cell-intrinsic/cell-extrinsic roles in cancer, reveal convergent cooperativity in a metastatic miRNA network, identify ApoE as an anti-angiogenic and metastasis-suppressive factor, and uncover multiple prognostic miRNAs with synergistic combinatorial therapeutic potential in melanoma.

Researchers at the Uniformed Services University of the Health Sciences show that levels of endogenous REDD1 are highly expressed in differentiated hematopoietic cells in response to radiation, which might be associated with radiation tolerance of the cells⁴.

To further understand the mechanisms of radiation-induced damage in different cells, microRNA (miRNA)-arrays were performed using purified miRNAs from CD34+ and hFOB cells before and post-irradiation. Their data, published in the latest PLoS ONE, suggest that CD34+ and hFOB cells have different miRNA expression patterns after irradiation and miR-30c plays a key role in radiation-induced cell damage which might be through regulation of REDD1 expression.

1. Tumaneng K, Schlegelmilch K, Russell RC, Yimlamai D, Basnet H, Mahadevan N, Fitamant J, Bardeesy N, Camargo FD, Guan KL. (2012) YAP mediates crosstalk between the Hippo and PI(3)K-TOR pathways by suppressing PTEN via miR-29. Nat Cell Biol [Epub ahead of print]. [abstract]
2. Hu S, Wilson KD, Ghosh Z, Han L, Wang Y, Lan F, Ransohoff KJ, Wu JC. (2012) MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2. Stem Cells [Epub ahead of print]. [article]
3. Pencheva N, Tran H, Buss C, Huh D, Drobnjak M, Busam K, Tavazoie SF. (2012) Convergent Multi-miRNA Targeting of ApoE Drives LRP1/LRP8-Dependent Melanoma Metastasis and Angiogenesis. Cell [Epub ahead of print]. [abstract]
4. Li XH, Ha CT, Fu D, Xiao M. (2012) Micro-RNA30c Negatively Regulates REDD1 Expression in Human Hematopoietic and Osteoblast Cells after Gamma-Irradiation. PLoS One 7(11), e48700. [article]

Download this poster outlining some of the most significant discoveries.

Title: Recent Work & Current Methods in Cardiovascular microRNA Research

Date: Tuesday – 11/27/12

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America  – 1:00 Central Daylight Time

https://attendee.gotowebinar.com/register/6411779313555818752

Europe, Asia – 1:00 PM Greenwich Mean Time

https://attendee.gotowebinar.com/register/4181057137775080704

Presented by
Dr. Christoph Eicken

Head of Technical Services

LC Sciences LLC

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments in cardiovascular microRNA research
• Current methods for microRNA discovery and profiling
• Case studies and application examples

After registering, you will receive a confirmation email containing information about joining the webinar.
After completion of the webinar, you will receive an email containing a link to the archived webinar.

In part, they sought to describe in detail the binding sites that comprise the Bacillus thuringiensis tubC DNA centromere. The structure of the centromere is defined by the binding of its protein partner; hence, they were able to precisely define tubC through the use of a protein-binding DNA aptamer microarray.

The researchers designed a microarray in which each experimental measurement consisted of a 24-bp double-stranded DNA sequence, formed by a hairpin stabilized through a tetraloop. This 24-base “window” was scanned through the plasmid in successive measurements; a 4-bp shift per measurement was used to conduct a coarse scan of a large area of pBtoxis, and single base pair shifts were then used for a high-resolution scan of the Bt tubC region. Bt TubR bound the array in only a few places, and binding was sustained reproducibly with the movement of the experimental window. The Bt tubC region was readily identified as the sole site of strong, sustained binding.

A Bt tubC microarray was probed with TubR. (A) An area of the Bt tubC microarray showing binding of Bt TubR (black) to sequences sampled at 4-bp intervals through pBtoxis (bp 122000 to 3100). The sequence runs from left to right, top to bottom. (B) Part of the microarray showing binding of Bt TubR (black) to sequences sampled at 1-bp intervals through part of Bt tubC (bp 126577 to 126530), the aligned sequences of the hairpins are shown, magenta indicating the consensus site.

The microarray revealed that Bt tubC in fact contains seven high-affinity binding sites, and in further experiments, the researchers demonstrate that the full region of binding sites is required for proper in vitro activity.

Bt tubC is composed of seven repeats, which bind TubR in a cooperative fashion. (A) Schematic comparing the tubZRC loci of Bt pBtoxis and Bm pBM400. (B) Illustration of the DNA hairpins produced on a microarray to sample the sequence of pBtoxis, and a schematic indicating how this window was scanned through a region of the plasmid sequence by successive single base pair movements. The variable window is shown in cyan, and all other base pairs in black. (C) Plot of the recorded signal for each microarray spot in a 1-bp scan over the region of Bt tubC (bp 126688 to 126496). Each point is plotted over the 12th bp of the 24-bp hairpin, with the sequence shown below. The assigned binding sites for Bt TubR are shown above, and the corresponding sequences are colored magenta below. The site(s) resulting in each peak have been annotated above the graph.

• Aylett CH, Löwe J. (2012) Superstructure of the centromeric complex of TubZRC plasmid partitioning systems. Proc Natl Acad Sci USA [Epub ahead of print]. [abstract]

In this study, an infectious clone from an epidemic ALV-J Chinese isolate designated HLJ09SH01 was constructed and rescued. The rescued virus was inoculated into specific-pathogen-free (SPF) layer chickens, and infected chickens produced tumors. Accumulating evidence shows that microRNAs (miRNAs) have a close relationship with tumorigenesis.

To gain more insight into the tumorigenesis of ALV-J, a miRNA microarray was performed by LC Sciences as part of an investigation of changes in host miRNA expression in a liver tumor from ALV-J infected chickens. The results showed that four miRNAs were significantly differentially expressed; these data were verified using real-time PCR. Bioinformatics analysis showed the differentially expressed miRNAs to be involved in some tumorigenesis-related signaling pathways, such as the MAPK signaling pathway and the Wnt signaling pathway, which may represent a possible signaling pathway that was involved in the ALV-J-induced tumorigenesis.

• Wang Q, Gao Y, Ji X, Qi X, Qin L, Gao H, Wang Y, Wang X. (2012) Differential expression of microRNAs in avian leukosis virus subgroup J-induced tumors. Veterinary Microbiology [Epub ahead of print]. [abstract]

Now, researchers at the University of Pittsburgh School of Medicine have developed a highly reproducible method that can simultaneously enrich a large number of CMs and cardiac SMCs and ECs from human induced pluripotent stem (iPS) cells with high purity.

Human multipotent cardiovascular progenitor cells were generated from human iPS cells, followed by selective differentiation of the multipotent cardiovascular progenitor cells into CMs, ECs, and SMCs. With further fluorescence-activated cell sorting, each of the three cardiovascular cell types could be enriched with high purity (>90%). These enriched cardiovascular cells exhibited specific gene expression signatures and normal functions when assayed both in vitro and in vivo. Moreover, CMs purified from iPS cells derived from a patient with LEOPARD syndrome, a disease characterized by cardiac hypertrophy, showed the expected up-regulated expression of genes associated with cardiac hypertrophy.

Overall, this technical advance provides the means for generating a renewable resource of pure human cardiovascular cells that can be used to dissect the mechanisms of human inherited heart disease and for the future development of drug and cell therapeutics for heart disease.

• Lin B, Kim J, Li Y, Pan H, Carvajal-Vergara X, Salama G, Cheng T, Li Y, Lo CW, Yang L. (2012) High-purity enrichment of functional cardiovascular cells from human iPS cells. Cardiovasc Res 95(3), 327-35. [abstract]

Visit us at Booth #1147and ask us about the latest developments in cardiovascular microRNA research.

.

Title: Recent Work & Current Methods in Cardiovascular microRNA Research

Date: Tuesday – 12/04/12

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America  – 1:00 Central Daylight Time

https://attendee.gotowebinar.com/register/6411779313555818752

Europe, Asia – 1:00 PM Greenwich Mean Time

https://attendee.gotowebinar.com/register/4181057137775080704

Presented by
Dr. Christoph Eicken

Head of Technical Services

LC Sciences LLC

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments in cardiovascular microRNA research
• Current methods for microRNA discovery and profiling
• Case studies and application examples

After registering, you will receive a confirmation email containing information about joining the webinar.
After completion of the webinar, you will receive an email containing a link to the archived webinar.

Previous Webinar links

This will be the 5th in a series of webinars covering the latest developments in microRNA research.  If interested, you can find links to recordings and slides from the previous two webinars.

Plant microRNA Webinar –

Cancer microRNA Webinar –

Endocrinology microRNA Webinar –

Neuroscience microRNA Webinar –

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) has been one of the successfully exploited baculoviruses, which is widely used as a bio-pesticide against major agricultural pests such as the cotton bollworm.

Researchers at the University of Queensland, Australia set out to determine how the bollworm’s immune system responds to and fights off the infection of the virus.

Deep sequencing of small RNA samples of infected bollworm performed by LC Sciences revealed a large number of small reads (∼20 nucleotides) mapped to the HaSNPV genome. The reads were mapped to certain open reading frames in the viral genome (hotspots) that are mostly structural and auxiliary late genes.

Subsequently, miRDeep was used to predict potential stem-loop structures in HaSNPV genome that may serve as pre-miRNAs. After mapping the small RNAs to the potential predicted stem loop structures, based on read counts, number of passenger strand, pairing in duplex and other parameters, it was determined that none of the stem-loop structures qualify as pre miRNAs.

After excluding the possibility of these small RNAs to be microRNAs, it was determined that Dicer-2, the main enzyme implicated in the RNAi response in insects, is involved in the generation of v-siRNAs. In Dicer-2, but not Dicer-1 silenced cells, higher transcript levels of the hotspot genes were detected and as a consequence the virus replicated more efficiently.

The results suggest that in insects, the RNA interference (RNAi) pathway plays a major role in anti-viral responses as shown against many RNA viruses. The response includes the cleavage of double stranded RNA genome or intermediates, produced during replication, into viral short interfering RNAs (v-siRNAs) which then degrade viral transcripts.

• Jayachandran B, Hussain M, Asgari S. (2012) RNA interference as a cellular defense mechanism against the DNA virus, baculovirus. J Virol [Epub ahead of print]. [abstract]

Download the presentation slides.

A team led by researchers at Tulane School of Medicine has now compared the expression of miRNAs in 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Their findings revealed a profound difference in miRNA profiles between 2-D and 3-D cultures of lung adenocarcinoma cells and a considerable portion of the differentially expressed miRNAs bear well-documented tumor-modulating activities. The 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in 3-D culture.

These results indicate that 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.

• Li C, Nguyen HT, Zhuang Y, Lin Z, Flemington EK, Zhuo Y, Kantrow SP, Morris GF, Sullivan DE, Shan B. (2012) Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures. Gene S0378-1119(12)01202-4. [abstract]

Date: Tuesday – 10/09/12

Please register for the webinar by selecting the presentation time that is most convenient for your location.

North, South America – 1:00 PM US Central Daylight Time
https://attendee.gotowebinar.com/register/3533891190071340288

Europe, Asia – 1:00 PM Greenwich Mean Time
https://attendee.gotowebinar.com/register/8037365473116295936

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments of microRNA research in the field of Neuroscience
• Current methods for microRNA discovery and profiling
• Neuroscience case studies and application examples

After registering, you will receive a confirmation email containing information about joining the webinar.

After completion of the webinar, you will receive an email containing a link to the archived webinar.

Previous Webinar links

This will be the 4th in a series of webinars covering the latest developments in microRNA research.  If interested, you can find links to recordings and slides from the previous two webinars.

Plant microRNA Webinar –

Cancer microRNA Webinar –

Endocrinology micrRNA Webinar –

The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA microarray analysis revealed that miR-29c was significantly downregulated in gastric cancer tissues relative to non-tumor gastric mucosae. Downregulation of miR-29c was associated with progression of gastric cancer, and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. Expression of the oncogene Mcl-1, a target of miR-29c, was significantly increased in gastric cancer tissues relative to non-tumor gastric mucosae.

Celecoxib (Brand name: Celebrex) – is in a class of nonsteroidal anti-inflammatory drugs (NSAIDs) pain relievers called COX-2 inhibitors.  It is used to relieve pain, tenderness, swelling and stiffness caused by various forms of arthritis.

The researchers found that treatment of human gastric cancer cells with celecoxib caused activation of miR-29c which induced suppression of Mcl-1 and apoptosis of the cancer cells. These results suggest that selective COX-2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR-29c.

•  Saito Y, Suzuki H, Imaeda H, Matsuzaki J, Hirata K, Tsugawa H, Hibino S, Kanai Y, Saito H, Hibi T. (2012) The tumor suppressor microRNA-29c is downregulated and restored by celecoxib in human gastric cancer cells. Int J Cancer [Epub ahead of print]. http://onlinelibrary.wiley.com/doi/10.1002/ijc.27862/abstract

We have some important information regarding our news blog feed.  Google has decided to shut down Feedburner.  This is the service we were using to deliver our news posts and manage our email subscriptions.  They will discontinue this service as of Oct 20th, 2012 so we have changed over to WordPress feeds and Mailchimp email subscriptions.

If you had subscribed to LC Sciences’ News Blog in an RSS reader, you will need to update your subscription from:

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If you had subscribed to LC Sciences’ News Blog by email, there is no action required.  You will continue to receive emails each time we make a post to the blog.  The email will have a slightly different look, as you can see.  We think it looks much nicer.  What do you think?

Sincerely,

The LC Sciences News Team

American Heart Association – Scientific Sessions
Nov 3-7, 2012 – Los Angeles, CA

ASCB 2012 – Annual Meeting of the American Society for Cell Biology
Dec 15-19, 2012 – San Francisco, CA

Rice University – Dept. of Biochemistry & Cell Biology

Date: Monday, September 17, 2012

Time: 9:30 AM
Location: Rice University
Address: George Brown Hall, (Campus Map), Outside, in the Breezeway, Houston, TX 77251-1892

(More Info…)

Texas Medical Center

Date: Wednesday, September 19, 2012

Time: 9:00 AM
Location: Trevisio Ballroom – The Commons Building
Address: 6550 Bertner Avenue, Houston, TX 77030 (Campus Map)

(More Info…)

Current standard microRNA arrays exist only for model species such as Human, Mouse, and Rat due to the limited sequence information available for non-model and exotic species. Model species arrays are designed based on a limited number of verified sequences deposited in public databases such as miRBase. As a result, researchers have been forced to use the array design /sequences of similar species. However, it is often very difficult to make sense out of these data: what is a perfect match, what is a mismatch signal, etc. Even with ability to make custom arrays for other species, prior sequence knowledge is required and one is still limited to known sequences that exist in public databases, such as miRBase.

Seq-Array offers a potential solution to high-throughput genome-wide microRNA profiling in species with limited or no microRNA sequence information available. This uniquely customized combination of the latest deep sequencing technology, advanced bioinformatics, and custom microarray leverages all these technologies to deliver a powerful dataset.

Recently developed microRNA sequencing technologies make it possible to identify and quantitatively decode the entire population of microRNAs in any sample, regardless of species or sample origin. Because no hybridization is involved, there is no need to design or predict any probes based on prior sequence knowledge, secondary structure, or sequence length information, and isomirs (slight variations of mature miR sequences) are all sequenced equally.

Advanced, bioinformatics filter the raw sequencing reads, classify all of the mappable reads, and create a microRNA “atlas” of all microRNAs in the sample.  From this microRNA atlas, a custom microarray is created for high-throughput screening of all samples in the experiment designed to detect exactly what each researcher is looking for as well as to validate any newly discovered microRNA sequences.

Application Note

MicroRNAs have a substantial impact on plant development and increasing evidence indicates that microRNAs play important roles in plant embryo development. Hybrid yellow poplar (Liriodendron tulipifera×L. chinense) is a fast-growing hardwood tree that has much agricultural value and occupies a “basal angiosperm” position in plant phylogeny making it an ideal candidate for comparative studies of the evolutionary history of flowering plants. However, when researchers at the Key Laboratory of Forest Genetics and Gene Engineering at Nanjing Forestry University, China set out to study this species, they ran into common problems among plant researchers: the Liriodendron genome is not completely sequenced, the number of expressed sequence tags (ESTs) in the database is limited, only a few microRNA species in Liriodendron have been reported, and computational prediction is not an effective method for discovering microRNAs that exist during somatic embryogenesis in hybrid yellow poplar. Their solution was a combination of next-gen sequencing and microarray hybridization which enabled the discovery of 232 candidate conserved microRNAs from 61 microRNA families and 273 candidate species-specific microRNAs in hybrid yellow poplar.

• Li T, Chen J, Qiu S, Zhang Y, Wang P, Yang L, Lu Y, Shi J. (2012) Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar. PLoS ONE 7(8), e43451. [article]

Date: Tuesday – 9/11/12

Please register for the webinar by selecting the presentation time that is most convenient for your timezone.

Western Hemisphere – 1:00 PM US Central Daylight Time

https://attendee.gotowebinar.com/register/7550994304927169280

Eastern Hemisphere – 1:00 PM Greenwich Mean Time

https://attendee.gotowebinar.com/register/1299179985486020864

Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments of microRNA research in the field of endocrinology
• Current methods for microRNA discovery and profiling
• Case studies and application examples

After registering, you will receive a confirmation email containing information about joining the webinar.

After completion of the webinar, you will receive an email containing a link to the archived webinar.

Previous Webinar links

This will be the 3rd in a series of webinars covering the latest developments in microRNA research.  If interested, you can find links to recordings and slides from the previous two webinars.

Plant microRNA Webinar –

Cancer microRNA Webinar –

In plants, miRNAs not only post-transcriptionally regulate their own targets but also interact with each other in regulatory networks to affect many aspects of development, such as growth, development and responses to biotic and abiotic stresses. Hundreds of miRNAs have been identified in higher plants by direct cloning or more recently by next-gen sequencing. To determine the function of these miRNAs we must first identify their targets.

Originally, plant miRNA targets have been studied via computational prediction, which is based on either perfect or near-perfect sequence complementarity between miRNA and the target mRNA or sequence conservation among different species. However, target prediction is very challenging, especially when a high level of mismatches exists in miRNA:target pairing.

Recently, a new method called degradome sequencing, which combines high-throughput RNA sequencing with bioinformatic tools, has-been successfully established to screen for miRNA targets in plants. Using degradome sequencing, many of the previously validated and predicted targets of miRNAs have been verified indicating that it is an efficient strategy to identify smRNA targets on a large scale in plants.

Degradome sequencing reveals miRNA targets by globally identifying the remnants of small RNA-directed target cleavage by sequencing the 5′ ends of uncapped RNAs. Sequencing reads are mapped to mRNAs and the 5′ terminal nucleotide of miRNA-cleaved mRNA fragments corresponds to the nucleotide that is complementary to the 10th nucleotide of the miRNA. Therefore, the cleaved RNA targets have distinct peaks in the degradome sequence reads at the predicted cleavage site relative to other regions of the transcript. Confirmed miRNA targets are presented in the form of target plots (t-plots).

Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases. However, unlike Arabidopsis and other plants, much less is known about its miRNAs and their targets. Now, researchers at the Chinese Academy of Agricultural Sciences, Beijing have used Illumina high-throughput sequencing analysis of small RNAs as well as degradome sequencing of B. napus to identify novel miRNAs and miRNA targets.

Forty-one conserved B. napus miRNAs and 62candidate novel B. napus-specific miRNAs were identified through small RNA sequencing and further verified by real-time RT-PCR.

A total of 33 non-redundant target ESTs for 25conserved miRNAs, and 19 non-redundant target ESTs for 17 B. napus-specific miRNAs were identified through degradome sequencing and verified by RNA ligase-mediated 5’RACE mapping.

This study describes large scale sequencing and identification of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.

• Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ, Sun J, Fan YL, Wang L. (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421. [article]

LC Sciences offers comprehensive services for both small RNA sequencing and degradome sequencing using the Illumina next-gen sequencing platform and in-house developed bioinformatics.

A team led by researchers at Sichuan Agricultural University has performed next-gen sequencing studies both on human and porcine miRNAs in breast milk exosomes.

They analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) and found that immune related miRNAs are present and enriched in breast milk exosomes¹. In Human breast milk the team found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes².

In both studies, the researchers demonstrated that, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions.

It is, therefore, tempting to speculate that these immune related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant’s immune system.

1. Gu Y, Li M, Wang T, Liang Y, Zhong Z, Wang X, Zhou Q, Chen L, Lang Q, He Z, Chen X, Gong J, Gao X, Li X, Lv X. (2012) Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes. PLoS ONE 7(8): e43691. [article]
2. Zhou Q, Li M, Wang X, Li Q, Wang T, Zhu Q, Zhou X, Wang X, Gao X, Li X. (2012) Immune-related microRNAs are abundant in breast milk exosomes. Int J Biol Sc 8(1), 118-23. [article]

Traditional applications of short, synthetic oligos in recombinant DNA technology include the introduction of restriction enzyme recognition sites for cloning, mutagenesis of a DNA segment, priming of PCR amplifications, and pasting together recombinant molecules. Today, the large scale and relatively low cost of short oligo synthesis with new oligo assembly techniques has resulted in replacing classic construct engineering by DNA synthesis, now frequently outsourced to commercial providers.

Most DNA synthesizers available in the market use phosphoramidite chemistry to elongate polystyrene- or controlled pore glass-bound oligos in the 3′ → 5′ direction ( [Caruthers, 1985], [Caruthers et al., 1983] and [Caruthers et al., 1987]). Certain characteristics of this scheme hinder its use in genome-scale applications. The pace of cost-reduction of column-based oligo synthesis seems to be slowing down, and the major part of oligo stocks is mostly wasted (more than is required) even when purchased in the smallest possible scale (Gibson et al., 2010b). In addition, accidental deletions, insertions, and depurinations may strongly reduce the fidelity of oligos, and consequently limit the length of robust synthesis (Hughes et al., 2011). Developments are, however, already on the way to control depurination (a common side reaction), allowing the reliable synthesis of oligos of 150 nt or longer ( [LeProust et al., 2010] and [Sierzchala et al., 2003]), but the most promising ways of decreasing costs and increasing throughput are microarray-based synthesis and microfluidics.

Microarray-based synthesis uses basically the same chemistries, but significantly reduces reagent-requirement and waste. At present, three different technologies are used for selective elongation of oligo chains in parallel: photolithography, the light-directed control of certain synthesis steps ( [Fodor et al., 1991], [Barone et al., 2001], [Gao et al., 2001] and [Singh-Gasson et al., 1999]), inkjet printing, the confinement of synthesis to discrete microdroplets ( [Blanchard et al., 1996] and [Hughes et al., 2001]), and electrochemically directed synthesis ( [Chow et al., 2009] and [Egeland and Southern, 2005]). Microchip synthesis can produce a vast number (up to 4.2 million) of different oligos at fractional costs (0.001 cent/nt (Carr and Church, 2009) vs. 10 cents/nt (Cheong et al., 2010)).

• Fehér T, Burland V, Pósfai G. (2012) In the fast lane: large-scale bacterial genome engineering. J Biotechnol 160(1-2), 72-9. [abstract]

LC Sciences’ microfluidic µParaflo^® system (based on the technology developed by Gao et al., 2001) enables the synthesis of OligoMix® for syntheic biology applications.

Deleting Sequences

• Misannotated sequences are deleted – next-gen sequencing data will enable more of this
• Cases of duplicate entries mapping to a single genomic locus are cleaned up – some enabled by new genome assembly releases
• Ex – Drosophila pseudoobscura V18 – 211 database entries, V19 – 210 database entries.

Renaming Sequences

• The process of retiring the miR/miR* nomenclature, in favour of the -5p/-3p nomenclature began with version 17 of the database.
• Version 17 – Drosophila sequences renamed.
• Version 18 – Human, Mouse, and Zebrafish sequences renamed.
• Version 19 – the miR* nomenclature is finally retired for all species.
• The miRBase curators note that the names are meant to be useful, but are not formally stable. They recommend that sequence names shouldn’t be used to convey complex information. Instead they recommend to use miRNA accession numbers, which do remain stable between releases, or you can always quote the sequence to be truly unambiguous. (miRBase Blog post regarding naming)

Adding Sequences

• Version 19 includes 3171 new hairpins and 3625 novel mature products.  That’s an 18% increase in the database size in a single update.

Because the rate of increase in size of the database is accelerating, it is making it more and more difficult for array manufacturers (that use the miRBase sequence database for probe content) to keep their microarrays up-to-date with the latest sequence information.  Often, pre-made arrays, sitting on the shelf, contain only a fraction of the known content for a particular species.

In comparison, LC Sciences’ microRNA microarrays are produced only as needed. This is made possible by our µParaflo® Biochip Technology – a high performance microfluidic custom microarray platform, enables on demand synthesis of microarrays.

Our microRNA arrays cover all species for which sequence data are available in the miRBase Sequence Database and the Plant MicroRNA Database.

Comparison of miRBase Versions

A comparison of the array versions available from various suppliers shows a significant amount of miRNA expression information is missed when using these out of date arrays for miRNA expression profiling analysis.

Comparison of miRBase Coverage

San Diego Regenerative Medicine Institute

Understanding the much more complex human embryonic development has been hindered by the restriction on human embryonic and fetal materials as well as the limited availability of human cell types and tissues for study. In particular, there is a fundamental gap in our knowledge regarding the molecular networks and pathways underlying human embryonic development. The enormous diversity of human somatic cell types and the highest order of complexity of human genomes, cells, tissues, and organs among all the eukaryotes pose a big challenge for characterizing, identifying, and validating functional elements in human embryonic development in a comprehensive manner. Many of the biological pathways and mechanisms of lower-organism or animal model systems do not reflect the complexity of humans and have little implications for the prevention and cure of human diseases in the clinical setting. As a result of lacking a readily available human embryonic model system, the mainstream of biomedical sciences is becoming increasingly detached from its ultimate goal of improving human health.

We recently reported that pluripotent hESCs maintained under a defined platform can be uniformly converted into a cardiac or neural lineage by small molecule induction, which enables lineage-specific differentiation direct from the pluripotent state of hESCs and opens the door to investigate human embryonic development using in vitro cellular model systems.MicroRNA (miRNA) expression profiling using microarrays is a powerful high-throughput tool capable of monitoring the regulatory networks of the entire genome and identifying functional elements in development and disorders. Recently advances in human miRNA expression microarrays have provided powerful genome-wide, high-throughput, and high resolution approaches that will lead to great advances in our understanding of the global phenomena of developmental processes. To identify mechanisms of small molecule induced lineage-specification of pluripotent hESCs, in this study, we compared the expression and intracellular distribution patterns of a set of cardinal chromatin modifiers in pluripotent hESCs, nicotinamide (NAM)-induced cardiomesodermal cells, and retinoic acid (RA)-induced neuroectodermal cells. Further, genome-scale profiling of miRNA differential expression patterns was used to monitor the regulatory networks of the entire genome and identify the development-initiating miRNAs in hESC cardiac and neural lineage-specification. We found that NAM induced nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing, while RA induced silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family to high levels. Genome-scale miRNA profiling indentified that a unique set of pluripotence-associated miRNAs was down-regulated, while novel sets of distinct cardiac- and neural-driving miRNAs were up-regulated upon the induction of lineage-specification direct from the pluripotent state of hESCs. These findings suggest that a predominant epigenetic mechanism via SIRT1-mediated global chromatin silencing governs NAM-induced hESC cardiac fate determination, while a predominant genetic mechanism via silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family governs RA-induced hESC neural fate determination. This study provides critical insight into the earliest events in human embryogenesis as well as offers means for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies. This original research article of Dr. Parsons supported by the National Institutes of Health was published in The International Open Access Journal of Stem Cell Research & Therapy

• Parsons XH. (2012) MicroRNA Profiling Reveals Distinct Mechanisms Governing Cardiac and Neural Lineage-Specification of Pluripotent Human Embryonic Stem Cells. J Stem Cell Res 2:124. [article]

(Read more about this research at: San Diego Regenerative Medicine Institute/News)

Read more about this update.

µParaflo^® Technology enables on demand synthesis of microarrays. That means this new probe content is immediately available on all of our standard or custom microRNA microarrays.

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. Although the viral receptor is an important factor for viral infectivity, other factors such as RNA interference (RNAi) can play an important role in antiviral responses and miRNA in particular may play an essential role in viral replication after host cell entry. It has been demonstrated that virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded miRNA species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates.

Researchers at the University of British Columbia, Canada, are studying the mechanisms by which miRNA-203 enhances Coxsackievirus B3 replication¹. Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. To identify miRNAs involved in CVB3 pathogenesis, they performed microarray analysis of miRNAs using CVB3-infected murine hearts and identified miR-203 as one of the most upregulated candidates. They identified zinc finger protein-148 (ZFP-148), a transcription factor, as a novel target of miR-203. Ectopic expression of miR-203 downregulated ZFP-148 translation, increased cell viability and subsequently enhanced CVB3 replication. Silencing of ZFP-148 by siRNA showed similar effects on CVB3 replication. Finally, analyses of the signalling cascade downstream of ZFP-148 revealed that miR-203-induced suppression of ZFP-148 differentially regulated the expression of prosurvival and proapoptotic genes of the Bcl-2 family proteins as well as the cell cycle regulators.

Another group of researchers, at Jilin University, China, found a link between viral infection, miRNA, and the Bcl-2 family of anti-apoptotic factor proteins². They found that several apoptosis-associated miRNAs were stimulated in influenza A virus-infected A549 cells by miRNA array analysis. Within those, miR-29c was significantly up-regulated. Luciferase assay confirmed the in silico predicted targeting of BCL2L2 3’UTR by miR-29c. Functional overexpression of miR-29c with miR-29c precursor inhibited BCL2L2 protein expression. Tansfection of miR-29c inhibitor abolished both suppression of BCL2L2 protein expression and A549 cells apoptosis induced by influenza A virus. These findings indicate that miR-29c-mediated BCL2L2 suppression is involved in influenza virus-induced apoptosis.

Also studying the effects of Influenza A Virus Infection on miRNA expression, a group from Northwestern University, United States used microarray profiling to show that several endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains³. Known antiviral response pathways, including Toll-like receptor (TLR), RIG-I-like receptor (RLR), and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems.

1. Hemida MG, Ye X, Zhang HM, Hanson PJ, Liu Z, McManus BM, Yang D. (2012) MicroRNA-203 enhances Coxsackievirus B3 replication through targeting zinc finger protein-148. Cell Mol Life Sci. 2012 Jul 29. [Epub ahead of print]. [abstract]
2. Guan Z, Shi N, Song Y, Zhang X, Zhang M, Duan M. (2012) Induction of the cellular microRNA-29c by influenza virus contributes to virus-mediated apoptosis through repression of antiapoptotic factors BCL2L2. Biochem Biophys Res Commun [Epub ahead of print]. [abstract]
3. Buggele WA, Johnson KE, Horvath CM. (2012) Influenza A Virus Infection of Human Respiratory Cells Induces Primary MicroRNA Expression. J Biol Chem [Epub ahead of print]. [article]

miRBase 19 is now available, brought to you from the Benasque RNA meeting in the sunny Pyrenees, and with a slightly larger time gap than usual. In that extended time, we have added more than the usual number of new sequences — 3171 new hairpins and 3625 novel mature products, bringing the totals to 21264 and 25141 respectively in 193 species. As always, the full README file is available on the FTP site, along with downloadable files containing all data in various formats.

We have spent some time deleting misannotated sequences, and the deep sequencing read views will allow us to focus more on this — 133 entries are removed in this release, many from the rice miRNA complement. We have also cleaned-up a number of cases of duplicate entries mapping to a single genomic locus (some prompted by new genome assembly releases) and rationalised many miRNA names. This is therefore a good time to remind you that the names are meant to be useful, but are not formally stable, and shouldn’t be used to convey complex information. The miRNA accession numbers *do* remain stable between releases, and of course, you can always quote the sequence to be truly unambiguous.

In this release, the miR* nomenclature is finally retired for all species, as previously promised. For every hairpin and mature sequence, all IDs that have previously been used in miRBase are now visible on the entry pages, and are downloadable in bulk from the FTP site.

At the time of writing, we have not added new deep sequencing datasets to the read view pages — however, a decent sized update to that section will be coming along shortly, together with an announcement here.

As always, comments, questions, abuse, praise all welcome here or by email.

• Buggele WA, Johnson KE, Horvath CM. (2012) Influenza A Virus Infection of Human Respiratory Cells Induces Primary MicroRNA Expression. J Biol Chem [Epub ahead of print]. [article]

Going beyond heuristics, emerging algorithms may enable automated design of RNAs with nucleotide-level accuracy but, as illustrated on a recent RNA square design, are not yet fully predictive. Looking ahead, technological advances in RNA synthesis and interrogation are poised to radically accelerate the discovery and stringent testing of design methods.

Perspective: an impending acceleration

In recent years, RNA designers have leveraged simple rules for secondary structure formation, the modularity of small RNA motifs, and three-dimensional closure to produce devices and nanostructures of growing complexity.

It is particularly exciting to see these designs deployed into biological systems, suggesting future routes to biomedical devices that sense and perhaps correct cellular dysfunction. We are optimistic about the development of more quantitative and predictive theories for RNA structure design and eventually RNA functional design due to the explosion of RNA data expected in upcoming years.

The cycle of RNA design and testing can be short, especially compared to protein engineering — arbitrary RNA sequences up to hundreds of nucleotides in length are experimentally straightforward to synthesize, purify, interrogate, and evolve on the timescale of days. Current technologies offer the parallel synthesis of thousands of arbitrary DNA templates (Figure 4a and b), which can be transcribed into RNA in vitro and, in principle, in vivo (Figure 4c). Further, multiplexed single-nucleotide- resolution chemical mapping and elegant selection strategies can provide information-rich data on all of these molecules’ structures and functions, using deep-sequencing platforms with turn-around times of hours (Figure 4d). The key challenges will then be to distribute and accurately analyze these data, to update RNA modeling algorithms, and to feed back these insights into the next rounds of synthesis and mapping.

The result, a massively parallel design cycle, would offer unprecedented opportunities for squaring theory with practice in RNA design.

• Bida J, Das R. (2012) Squaring theory with practice in RNA design. Curr Opin Struct Biol [Epub ahead of print]. [article]

• LC Sciences In The Press
• Mother’s Diet Affects Fetuses’ microRNA Expression
• microRNAs as Cryoprotectants
• LC Sciences Begins Webinar Series
• Seq-Array^SM Leverages the Power of both NGS and Microarray for Plant microRNA Studies
• Recent Customer Publications
• Recent Posters Presented by LC Sciences

A group of Canadian researchers hypothesized that microRNAs (miRNAs), small non-coding transcripts that bind to mRNA, could play a role in the global regulation of energy-expensive mRNA translation in frozen insects and would be modulated at subzero temperatures.

Expression levels of miRNA species were evaluated in control (5°C) and frozen (-15°C) goldenrod gall fly larvae, Eurosta solidaginis, using LC Sciences’ miRNA microarray service. Levels of several miRNAs were significantly reduced in frozen larvae whereas others rose significantly in frozen larvae. Target prediction for two of the differentially regulated miRNAs, miR-277-3p and miR-284, revealed potential regulation of transcripts involved in translation and the Krebs cycle.

These data constitute the first report that differential expression of miRNAs occurs in a freeze tolerant insect and suggest a mechanism for reversible gene regulation during prolonged periods of freezing over the winter months, a mechanism that can be rapidly reversed to allow renewed translation of mRNA when temperatures rise and insects thaw.

• Courteau LA, Storey KB, Morin PJ. (2012) Differential expression of microRNA species in a freeze tolerant insect, Eurosta solidaginis. Cryobiology [Epub ahead of print]. [abstract]

A team led by researchers at West Virginia University set out to characterize the egg miRNA transcriptome and identify novel egg-predominant miRNAs in rainbow trout.

Highlights from the study:

They isolated RNA from mature unfertilized rainbow trout eggs and utilized LC Sciences’ Small RNA Sequencing Service to accomplish this goal.

• The massive sequencing produced 24,621,741 quality reads
• 266 known miRNAs were identified and 230 putatively novel miRNAs were predicted
• The most abundantly known miRNAs are let-7 and miR-21
• Other known miRNAs which are abundantly present in eggs include miR-24, miR-202, miR-148, miR-30, miR-10, miR-146, miR-25, and miR-143

They followed the sequencing project with real-time PCR validation.

• Used cDNAs derived from 10 different tissues
• Validated 87 out of 90 selected putative miRNAs
• Identified three novel miRNAs predominantly expressed in rainbow trout eggs.
• Each of these novel egg-predominant miRNAs is predicted to target a significant number of genes
• Demonstrated low expression of a selected number of target genes in eggs relative to liver and muscle tissues

This study represents the first complete survey of miRNAs in fish eggs and provides a starting point for future studies aimed at understanding the roles of miRNAs in controlling egg quality and early embryogenesis in rainbow trout.

• Ma H, Hostuttler M, Wei H, Rexroad CE III, Yao J (2012) Characterization of the Rainbow Trout Egg MicroRNA Transcriptome. PLoS ONE 7(6): e39649. [article]

²The nature of host-virus interactions in hepatitis B virus infection is incompletely understood. Since soluble factors, e.g., cytokines and metals, may exacerbate liver injury in chronic hepatitis, we considered that defining the effects of receptor-mediated signaling upon viral replication will be significant. Consequently, we studied effects of iron or TGF-β-induced TGF-β/BMP signaling in the HepG2 2.2.15 cell model of hepatitis B virus replication.

³More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA) regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro. (microRNA Microarray Service)

miRNA expression profile during porcine follicular atresia.

Healthy (H), early atretic (EA), progressively atretic (PA) are representative regions of the Cy5 chip images. The images are displayed in pseudo-colors to expand the visual dynamic range. As the signal intensity increased from 1 to 65,535, the corresponding color changed from blue to green to yellow and red.

1. Yang G, Yang L, Zhao Z, Wang J, Zhang X (2012) Signature miRNAs Involved in the Innate Immunity of Invertebrates. PLoS ONE 7(6), e39015. [article]
2.  Park SO, Kumar M, Gupta S (2012) TGF-β and Iron Differently Alter HBV Replication in Human Hepatocytes through TGF-β/BMP Signaling and Cellular MicroRNA Expression. PLoS ONE 7(6), e39276.  [article]
3.  Lin F, Li R, Pan Zx, Zhou B, Yu Db, et al. (2012) miR-26b Promotes Granulosa Cell Apoptosis by Targeting ATM during Follicular Atresia in Porcine Ovary. PLoS ONE 7(6), e38640. [article]

Neuroscience, Oncology

Zhou J, Liu M, Jiang J, Qiao G, Lin S, Li H, Xie L, Zhuo R. (2012) Expression profile of miRNAs in Populus cathayana L. and Salix matsudana Koidz under salt stress. Mol Biol Rep [Epub ahead of print]. [abstract]

  Agriculture, Animal Science

Yang L, Lu X, Liu Y, Lv Z, Chen J, Yu W, Zhang Y, Nie Z. (2012) Expression analysis of miRNAs in BmN cells. Gene [Epub ahead of print]. [abstract]

  Cardivascular

Duval S, Massaro JM, Jaff MR, Boden WE, Alberts MJ, Califf RM, Eagle KA, D’Agostino RB, Pedley A, Fonarow GC, Murabito JM, Steg PG, Bhatt DL, Hirsch AT. (2012) An evidence-based score to detect prevalent peripheral artery disease (PAD). Vasc Med [Epub ahead of print]. [abstract]

Afonyushkin T, Oskolkova OV, Bochkov VN. (2012) Permissive role of miR-663 in induction of VEGF and activation of the ATF4 branch of unfolded protein response in endothelial cells by oxidized phospholipids. Atherosclerosis [Epub ahead of print]. [abstract]

  ]]> http://www.lcsciences.com/news/microrna-findings/feed 0 http://www.lcsciences.com/news/seq-array-leveraging-the-power-of-both-ngs-and-microarray-for-plant-microrna-studies http://www.lcsciences.com/news/seq-array-leveraging-the-power-of-both-ngs-and-microarray-for-plant-microrna-studies#comments Thu, 28 Jun 2012 17:56:00 +0000 admin http://www.lcsciences.com/news/?p=1419 A study led by researchers at Nanjing Agricultural University, China provides the first small RNA expression analysis throughout the entire grain filling phase in an indica rice cultivar.

MicroRNAs (miRNAs) modulate gene expression in different tissues and at diverse developmental stages in plants. In this study, the researchers used a Seq-Array approach to first, identify novel miRNAs in indica rice by small RNA sequencing and then, to study their expression patterns during all stages of grain development by custom microarray.

Using LC Sciences microRNA discovery and profiling services, sequenced small RNAs from all stages of grain development and discovered 11 potential novel miRNAs based on the presence of their miRNA*s. A total of 21 conserved and 91 non-conserved miRNA families were found in developing indica grains.

Expression patterns of these identified miRNAs were analyzed using customized miRNA chips. Comparison between the three phases of grain filling revealed that these miRNAs and their targets may be involved in diverse pathways, which may also be conserved in other cereal plants. The results showed that during the filling phase about half of the detected miRNAs were up-regulated, whereas the remainder were down-regulated. Predicted targets of differentially expressed miRNAs may participate in carbohydrate metabolism, hormone signaling and pathways associated with seed maturity.

The complexity of these miRNAs and their targets and interactions require further study to obtain a better understanding of the molecular mechanisms underlying grain development.

1. Lan Y, Su N, Shen Y, Zhang R, Wu F, Cheng Z, Wang J, Zhang X, Guo X, Lei C, Wang J, Jiang L, Mao L, Wan J. (2012) Identification of novel MiRNAs and MiRNA expression profiling during grain development in indica rice. BMC Genomics 13(1), 264. [Epub ahead of print]. [article]

Title: Recent Work & Current Methods in Cancer microRNA Research

Date: Wed 7/11/12

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Webinar Outline

• Brief review of microRNA basics: history, biogenesis, function
• Recent developments of microRNA research in the field of cancer genomics
• Current methods for microRNA discovery and profiling
• Case studies and application examples

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After completion of the webinar, you will receive an email containing a link to the archived webinar.

1

 Ammopiptanthus mongolicus is an ecologically important plant species in Mid-Asia desert and exhibits substantial tolerance to drought condition. Insufficient transcriptomic and genomic data in public databases has limited our understanding of the molecular mechanism underlying the stress tolerance of A. mongolicus. They used 454 pyrosequencing to identify assembled sequences representing approximately 9,771 proteins in PlantGDB. The 29,056 unique sequences in this 454EST collection represent a major transcriptomic level resource for A. mongolicus, and will be useful for further functional genomics study in Ammopiptanthus genus. The thousands of SSR markers predicted in our 454 ESTs should facilitate population genomic studies in Ammopiptanthus. The potential drought stress related transcripts identified in this study provide a good start for further investigation into the drought adaptation in Ammopiptanthus.

 ²A study led by researchers at Nanjing Agricultural University, China provides the first small RNA expression analysis throughout the entire grain filling phase in an indica rice cultivar.

Using LC Sciences microRNA discovery and profiling services, they discovered 11 potential novel miRNAs based on the presence of their miRNA*s. A total of 21 conserved and 91 non-conserved miRNA families were found in developing indica grains. Expression patterns of these identified miRNAs were analyzed using customized miRNA chips. Their small RNA sequencing and chip analysis enlarged the rice miRNA repertoire and confirmed the existence of most conserved, and nearly half of the non-conserved, rice miRNAs in developing grains. Comparison between the three phases of grain filling revealed that these miRNAs and their targets may be involved in diverse pathways, which may also be conserved in other cereal plants. The complexity of these miRNAs and their targets and interactions require further study to obtain a better understanding of the molecular mechanisms underlying grain development.

1. Zhou Y, Gao F, Liu R, Feng J, Li H, (2012) De novo sequencing and analysis of root transcriptome using 454 pyrosequencing to discover putative genes associated with drought tolerance in Ammopiptanthus mongolicus. BMC Genomics 13(1), 266. [article]
2. Lan Y, Su N, Shen Y, Zhang R, Wu F, Cheng Z, Wang J, Zhang X, Guo X, Lei C, Wang J, Jiang L, Mao L, Wan J. (2012) Identification of novel MiRNAs and MiRNA expression profiling during grain development in indica rice. BMC Genomics 13(1), 264. [Epub ahead of print]. [article]

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