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Happy Thanksgiving from LC Sciences.

Researchers at the University of Texas Southwestern Medical Center, Dallas used microarray analysis to compare miRNA expression patterns of primary rat cardiomyocytes with different levels of myocardin-related transcription factors (MRTFs). MRTFs associate with serum response factor (SRF) to drive the expression of actin and other cytoskeletal protein genes. miR-145 and miR-143 were among the most strongly upregulated miRNAs in response to MRTFs, as revealed by microarray and confirmed by real-time PCR. Further study revealed that (SRF) controls the expression of miR-143 and miR-145 in smooth muscle cells, and these miRNAs, in turn, feed back to control the expression and function of multiple components of the cytoskeleton and the SRF regulatory network. This study adds to a growing body of work demonstrating the roles of miRNAs in regulating changes in gene expression and cell functions in response to injury and stress and further underscores their potential as therapeutic targets.

Xin M, Small EM, Sutherland LB, Qi X, McAnally J, Plato CF, Richardson JA, Bassel-Duby R, Olson EN.  (2009) MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury.  Genes Dev 23(18),2166-178. [abstract]

Quantitative mapping of substrate specificity for protein kinase Src on a microarray
Chip Design

• PKS – peptide microarray
• 27 known protein tyrosine kinase substrates and their sequence variants
• All peptides on the chip contain 8-10 redundancies.
• Positive controls – synthetic phosphopeptides (pY phosphotyrosine incorporated by synthesis)
• Negative controls – Ala substitution of Tyr at the phosphorylation site
• Src kinase, p60c-src (Invitrogen)

Assay

• Sample – Src kinase, p60c-src (Invitrogen)
• Enzyme concentration – 0.5 mg/ml
• Volume – 50 µl
• Reaction time – 30 minutes or longer

Detection

• Fluorescent dye is used to specifically stain phosphate groups (pS, pT, and pY).
• Only the phosphate group of the phosphopeptides will be specifically stained by the fluorescent dye.  No need for antibodies which are known to suffer from non-specific or weak binding problems. If specially request, antibody detection can be applied.

Results

• Figure 1 – Fluorescent image (inverted) of 4K kinase profiling microarray
• (A) Image of a subset of four replicates of the I(Y/A)GEF, pY and Y sequences – The result shows that IYGEF is phosphorylated.
• (B) Image of the YVPM (column 1) and the YEEIP (column 2) related sequences in double replicates – Among these, YEE and YEEI are phosphorylated by the Src kinase used, and YEEIP is a substrate of lower reactivitiy, and YVPM has the lowest reactivity.  Their results are consistent with the literature information.

Figure 1

Figure 2 – Plot of the relative phosphorylation efficiency of each Y-containing peptide

• The phosphorylating efficiencies of YEEI and IYGEF surpassed 90% of the synthetic pYEEI or pYGEF.
• Except YLEL, Src kinase selected peptides with Glu, Asp or amino acids with small side chains (Gly or Ala) at the P+1 position. (ratio>30%, a line placed)
• This was also demonstrated by Songyang’s paper (Songyang, Z and Cantley, L.C., (1995) Recognition and specificity in protein tyrosine kinase-mediated signaling, Trends in Biology Sciences, 20, 470-475).  [abstract]

• Relative Phosphorylation Efficiency:  fp% = (I_Y-I_A) / (I_pY-I_A)*100
• I_Y – signal intensity of phosphorylated Y-peptide
• I_pY – intensity of synthetic phosphopeptide
• I_A – intensity of Y>A substitution peptide as negative control

Researchers at the University of Queensland, School of Biological Sciences studying Heliothis virescens ascovirus (HvAV3e) and a Heliothis zea fat body cell line (HzFB), used a custom insect miRNA microarray and found that Hz-miR24 is differentially expressed in insect hosts following virus infection with an increase in its expression levels late in infection¹. A custom miRNA microarray from LC Sciences contained 9 replicates of all known insect miRNAs (miRBase Release 12.0; total of 288 insect miRNAs) in addition to custom miRNA probes from HzFB cells and Bombyx mori (total of 94 miRNAs). Experimental evidence demonstrated that Hz-miR24 downregulates ascoviral DNA-dependant RNA polymerase and its β subunit transcript levels late in infection.  The recent availability of complete genome sequences of ascoviruses has made these discoveries possible. These findings suggest that ascoviruses may manipulate host miRNAs that in turn regulate expression of their genes at specific time points after infection.

1. Hussain M, Asgari S.  (2009) Functional analysis of a cellular microRNA in insect host-ascovirus interaction.  J Virol [Epub ahead of print] [abstract]

ASCB 2009 – American Society for Cell Biology – Annual Meeting
Dec 5 – 9, 2009 – San Diego, CA       Visit LC Sciences at Booth #844

As the premier international meeting in the field of cell biology, the ASCB Annual Meeting is intended for scientists and students in academia, industry, government, and higher education. Over 100 scientific sessions and 3,500 poster presentations cover a variety of scientific areas within the discipline. With opportunities to learn about the latest research and network with peers, the ASCB Annual Meeting appeals to the diverse interests of the international cell biology community.

 

Plant & Animal Genomes XVIII Conference
Jan 9 – 13, 2010 – San Diego, CA     Visit LC Sciences at Booth #509

The Premier and Largest Ag-Genomics Meeting in the World

PAG XVIII will bring together the leading genetic scientists and researchers involved in plant and animal research and related areas. With over 30 countries represented, the Plant & Animal Genome Conference provides an established forum for the exchange of information internationally as well as domestically.

Many recent studies report that miRNAs control the key components of signaling pathways and thereby regulate related development processes in plants.  LC Sciences offers several options for study of plant microRNA.  In addition to our standard microarrays designed for all plants in the miRBase 14.0 (1,117 unique miRs) and the Plant MicroRNA Database 1.0 (5,690 unique miRs), our custom microfluidic platform enables the synthesis of made to order custom plant microarrays.

Researchers at the Laboratory of Tropic Biological Resources, Hainan University, China utilized a custom plant microRNA microarray from LC Sciences to study cellular development in coconut, a key plantation crop in the tropical parts of the world¹. The custom µparaflo® microfluidic chip contained 653 unique plant miRNAs of release version 10.1 (http://www.mirbase.org/), representing 877 miRNAs from 17 plant species. The 877 miRNAs comprised 154 from Arabidopsis thaliana, 115 from Oryza sativa, 187 from Physcomitrella patens, 100 from Populus trichocarpa, 43 from Zea mays and 278 from 12 other plant species.

Comparative analyses of the miRNA expression profiles between developmental stages of coconut endosperm showed that several miRNAs were differentially expressed in mature tissue. Differential expression was confirmed using real-time PCR. The contrasting expression patterns between the two developmental stages suggested that miRNAs play a role in tissue development and compound metabolism of coconut endosperm.

Li D, Zheng Y, Wan L, Zhu X, Wang Z. (2009) Differentially expressed microRNAs during solid endosperm development in coconut (Cocos nucifera L.). Scientia Horticulture 122(4), 666-69. [abstract]

LC Sciences now offers a comprehensive epitope mapping service for high throughput, high-resolution identification of epitopes and other protein-protein interactions.

Through the use of overlapping peptides as epitopes on a custom synthesized addressable peptide microarray (PepArray™), we can systematically screen thousands of sequences in a single experiment.  A proprietary microarray platform and advanced microfluidic technologies ensure quantitative measurements of binding events.

This combination of high-throughput capacity with quantitative measurement enables us to quickly and efficiently identify high affinity and high specificity target binding compounds.

Epitope mapping assays are applicable for immunological studies, vaccine development, and biosensor development.

• Map immunodominant region in an antigen
• Develop epitope-based vaccines
• Describe antibody binding in detail for regulatory or patent filings
• Determine the mechanism of a biological process

Researchers at the LSU Neuroscience Center have discovered a role for miRNA-146a in the evasion of Herpes simplex virus type-1 (HSV-1) from the complement system (a major first-line host defense mechanism), and the activation of key elements of the arachidonic acid cascade known to contribute to Alzheimer-type neuropathological change.

MicroRNA microarray revealed that human primary neural cells infected with HSV-1 (17syn +) showed upregulation of miRNA-146a, a brain-enriched microRNA that is associated with proinflammatory signaling in stressed brain cells and Alzheimer’s disease.

Hill JM, Zhao Y, Clement C, Neumann DM, Lukiw WJ.  (2009) HSV-1 infection of human brain cells induces miRNA-146a and Alzheimer-type inflammatory signaling.  Neuroreport  20(16), 1500-505.  [abstract]

Researchers at Heidelberg University used a combination of expression profiling of miRNAs and subsequent functional inhibitory screening in primary hippocampal neurons to identify miRNAs in the synaptodendritic compartment that function during synaptic development.  MicroRNA microarrays identified ten mature miRNAs that were enriched and four mature miRNAs that were strongly depleted in synaptosomes compared with whole forebrain. Northern blot analysis on selected miRNAs confirmed the microarray results.  Subsequent functional screening identified miR-138 as a negative regulator of dendritic spine size and revealed that mRNA encoding acyl-protein thioesterase 1 (APT1) is a miR-138 target in neurons.

Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S, Christensen M, Khudayberdiev S, Leuschner PF, Busch CJ, Kane C, Hübel K, Dekker F, Hedberg C, Rengarajan B, Drepper C, Waldmann H, Kauppinen S, Greenberg ME, Draguhn A, Rehmsmeier M, Martinez J, Schratt GM.  (2009) A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis.  Nat Cell Biol 11(6), 705-16.  [abstract]

Why synthesize thousands of oligonucleotides individually?

Why perform thousands of individual reactions while handling thousands of tubes?

When you can

Synthesize one OligoMix®.
Perform thousands of parallel (multiplex) reactions in a single tube.
Save time and money.
Be kind to our environment.
 
For instance; for a small genome of 5Mbp, 250,000 oligos of 40mers are required.  To synthesize thousands of oligo sequences individually would be expensive, equipment intensive, impractical.

Making thousands of oligonucleotides consumes a huge amount of solvents and chemical reagents which is environmentally-unfriendly, costly, and time consuming.  The synthesis per se would need 60,000 liters of solvents and cost $1M.  It would take a gene synthesis company a year to complete.

DNA synthesis of individual oligonucleotides is a major operation requiring significant up-front investment in synthesis instrumentation.  Significant personnel are required for time consuming synthesis and handling processes.

Performing multiplex applications quickly and accurately using synthetic oligonucleotides in individual tubes or plates requires sophisticated robotic instrumentation that is maintenance intensive and requires highly technical personnel.

Imagine if one person increases experimental throughput by 100-fold, one would need 100-times more glass and plastic-ware and 100-times more material consumption.  If there is no change in the way we do things, our research budget and space requirements would grow by 100-fold or more! 

Consider OligoMix® as a green alternative.

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