General

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are recently found to be negative regulators of gene expression in eukaryotic organisms. Newly synthesized primary miRNA transcripts (pri-miRNAs) are processed by RNase III like enzyme, Dicer, to generate ~70 to 100 nucleotide (nt) hairpin precursors (Pre-miRNAs). Pre-miRNAs which are further processed by another RNase III like enzyme, yield mature miRNAs, averaging 21-23 nt in length. miRNAs are incorporated into the RNA interference (RNAi) effector complex, RISC, and target specific messenger RNAs for translational repression or mRNA cleavage. MicroRNAs show distinct expression patterns in different organisms, cell development stages, and disease models. Therefore, miRNAs play an important role in regulating gene expression 1-3.

References:

1. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–-297 (2004).

2. Ambros, V. The functions of animal microRNAs. Nature 431, 350–-355 (2004).

3. He, L. & Hannon, G. J. MicroRNAs: small RNAs with a big role in gene regulation. Nature Rev. Genet. 5, 522–-531 (2004).

Microarray Design

These are not spotted arrays! Our microRNA microarray synthesis is based on a proprietary µParaflo® microfluidic chip technology developed in house. This flexible technology enables fast, on chip synthesis of microarrays when ordered. (vs. an off-the-shelf spotted array) Please see our µParaflo® technology support documents for further information.

LC Sciences offers the most comprehensive line of standard microRNA detection arrays – all species for which sequence data exist in the miRBase Sequence Database. Although the sequence database is being continually updated as new sequences are experimentally verified, the contents of our standard arrays are updated in synchronization with the miRBase. This synchronization is made possible by our flexible µParaflo® microfluidic chip technology

We also offer a NO-COST option to add up to 100 probes (of 25 nt or shorter) of your own design on each array. This option gives you the opportunity for discovering new small RNAs, adding internal controls, verifying hypothesis, or achieving any other enhancement that you would like to have.

We also offer totally custom arrays at a very reasonable cost. This service gives you total freedom for the choice of probes up to 3,748 sequences. For example, you can screen for new microRNAs by adding predicted mature microRNA sequences or performing sequence walks along certain sequences sections. You can combine microRNA sequences of different species to identify cross-species conservations, add controls for the detection of customer-added spiking RNA sequences, or add probes for the detection of siRNAs and/or other small non-coding RNAs.

Please talk to our customer support about your requirements. Also see the Technical Bulletin discussing probe selection.

miRBase is a sequence database that has been established by the Sanger Institute. Each entry in the microRNA Registry represents a predicted hairpin portion of a microRNA transcript (termed mir in the database), with information on the location and sequence of the mature microRNA sequence (termed miR).

The database was established with two broad aims:

The content of our microarrays was updated to miRBase Release 21 in July 2014.

We include a minimum of four redundancies of each miRNA probe on the single species arrays. For multi species arrays, the number of redundancies is reduced due to the increased number of probe sequences. Please contact us before ordering multi species arrays.

Each of our detection probes contains a coding segment and a spacer. The coding segment is a nucleotide sequence involving proprietary chemical modification for enhancing the detection of target transcripts. The spacer is a non-nucleotide molecule that extends the detection probe away from substrate and therefore further enhances the binding between the probe and the target. The length of the detection probes varies according to different targets.

Yes, the Tms of our detection probes are balanced. By varying the number of modified nucleotides in each probe, we adjust the Tm of that probe. Our array detection signals are more uniform due to balanced Tms.

No, we have done extensive studies and verified that these chemical modifications enhance weak signals without sacrificing binding specificity. We use specificity control probes on every chip and we show a perfect match to mismatch ratio of more than 10 for a spike-in control RNA sequence. Customers can easily examine the binding specificity of their array results by looking at control signals in the data they receive from us.

For single species arrays, we synthesize all sequences in a minimum of four replicates on the chip and our intra chip variability is very low. Further, because our chips are based on the µParaflo® technology, the spot uniformity is excellent both within the chip and from chip to chip. The decision of how many replicate chips to run is up to you. Generally, customers will run a single chip and make an assessment to determine if experimental design is OK or needs to be altered before multiple chips are run to validate results. If you think there might be variability in your sample, you may want to run multiple (3) arrays.  See the Technical Bulletin for more detail on experimental design for microarray experiments.

Probes on our arrays are synthesized using chemical reagents of the highest quality. Every array produced has to pass a rigorous QC process which involves the hybridization with two testing DNA oligos labeled with Cy3 and Cy5, respectively. There are 16 sets of control probes on each array for the production QC. Our QC criteria include a across-array uniformity at a spot-to-spot CV less than 15%, a minimum hybridization intensity at a predetermined testing oligo concentration, and a match to (single-base substitution) mismatch ratio of larger than 10.

We spike a 20 mer control RNA into each sample followed by labeling and hybridization. The control RNA has been computationally and experimentally verified not to cross-hybridize with the probes of any known miRNA transcript. On each array there are 16 sets of control probes spatially distributed across the array. Each set contains a perfect match and a single-base-substitution mismatch for the control RNA. Chip and assay qualities, such as uniformity and specificity, can be assessed by examining the signal intensities of these control probes. Typically, the CV of the spot-to-spot intensities of the perfect match probes is less than 15% and the intensity ratio of perfect match to mismatch probes is larger than 10.

Custom Sequences

Yes, you can add custom sequences to any of the standard miRNA arrays. Assuming that your longest custom sequence is less than 25 nt, there is no charge for adding up to100 additional sequences to the array.

If any of your sequences are longer than 25 nt (it doesn’t matter how many), there will be $5.25 charge for each additional nucleotide over 25 (count from the longest oligo).

There are 3,748 open features available for synthesis of customer sequences. Subtract the total number of standard sequences from this number to determine the number of custom sequences available. This number will vary slightly as each standard species array contains a different number of miRNA probes. Please talk to our customer support about your requirements.

If more than 100 custom sequences are needed, we treat the array as if it is a custom array. Contact us for details.

We recommend triplets although this will reduce the total number of sequences you can add to the array.

Sample Submission

Please complete a sample submission form prior to sending your sample to us. We request that you email the form to us beforehand AND place a copy of the form in the package along with your sample.

We cannot begin work on your array without a PO Number or Credit Card Number.

See our sample submission page for further information.

Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (0.5 ml or smaller PCR tubes can crack when frozen).

If you want to seal the tubes or hold them in a rack, please don’t use tape (it will crack when frozen), use parafilm.

Pack the sample with dry ice in a thermal insulated shipping box. Ship by overnight carrier for delivery the next day.

Note: To be on the safe side, we recommend to ship samples Monday – Wednesday. Do not ship samples on Friday as they will sit over the week-end and deteriorate. Wait until the following Monday to ship the package for Tuesday delivery.

Yes, we can return unused sample to you upon request. Please note that samples will be stored for a maximum of six months after your experiment has been completed.

Sample Preparation and Labeling

Currently we do not amplify the sample but instead we use a signal amplification strategy to detect small amounts of miRNA.

We use a proprietary labeling method which utilizes an affinity tag for signal amplification after miRNA hybridization to the chip.

Array Results

We can generally have data back to you about 2-3 weeks from the date we receive your total RNA sample. We also offer a 1-2 week rush service.

For each array, you will receive the original and processed microarray scan images, an array layout file, a raw intensity data file in Excel, a fully processed data file in Excel, and a list of up and down regulated transcripts that are called based on a statistical analysis. (dual sample arrays only)

Additionally, for each batch of samples, you will receive a Data Summary containing a catalog of data files, images of representative regions of corresponding arrays, and descriptions of specific features of the arrays.

The above files will be stored on a CD and will be delivered to you by express mail.

View and download Example Data Files.

Data Analysis

Yes, standard data analysis which consists of sample to sample comparison on the same array is included in the comprehensive service. In-depth analysis of samples across multiple arrays is also included.

Data will be corrected by subtracting background and normalized to the statistical median of all detectable transcripts. The data may also be normalized to one or a group of house-keeping genes that are added to the array as an option selected by customers. The data analysis also includes the calculation of p-values. Based on the p-value, a list of differentially expressed transcripts is produced. When a color reversal experiment is performed, two lists of differentially expressed transcripts from the two reverse labeled arrays are merged into one list. The differentially expressed transcripts having consistent calls on both arrays are grouped together in the merged list. You should focus mainly on this group of transcripts in your further studies.

We have in house software for routine array data processing that follows the common practices of DNA array data treatment.4-7 The data are processed in a MS Excel spreadsheet using a program routine that performs raw signal background subtraction using a local regression method (Xiaochuan Zhou, unpublished results; note that the photolithographically fabricated arrays do not have peripheral areas for background values)

Data normalization, using a cyclic LOWESS (Locally-weighted Regression) method8 is used to remove system related variations, such as sample amount variations, dye labeling bias, and signal gain differences between scanners, so that biological relevant variations can be faithfully revealed. Detected signals greater than background plus 3 times the standard deviation will be derived for each color channel; the mean and the co-variance (CV = stdev x100/replicate mean) of each probe having a detected signal will be calculated.

For two color experiments, the ratio (log transformed) of the two sets of detected signals, and p-values of the t-test, will be calculated. Differentially detected signals are generally accepted as true when the ratios of the p value is less than 0.01.

See the Technical Bulletin for a detailed description of the procedure we use to perform data analysis for the microRNA microarray service.

References:

4 Ball, C. A.; Sherlock, G.; Parkinson, H.; Rocca-Sera, P.; Brooksbank, C.; Causton, H. C.; Cavalieri, D.; Gaasterland, T.; Hingamp, P.; Holstege, F.; Ringwald, M.; Spellman, P.; Stoeckert, C. J., Jr.; Stewart, J. E.; Taylor, R.; Brazma, A.; Quackenbush, J. (2002) Standards for microarray data. Science 298, 539.

5. Quackenbush, J. (2001) Computational analysis of microarray data. Nature Rev. Genet. 2, 418-27.

6. Quackenbush, J. (2002) Microarray data normalization and transformation Nature Genet Suppl., 496-501.

7. Sturn, A.; Quackenbush, J.; Trajanoski, Z. (2002) Genesis: cluster analysis of microarray data. Bioinformatics 18, 207-8.

8 Bolstad, B. M.; Irizarry, R. A.; Astrand, M.; Speed, T. P.(2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-93

p-value is a statistic parameter that measures the similarity of Cy3 and Cy5 labeled transcripts. The smaller the p-value is, the less possible the Cy3 and Cy5 labeled transcripts are similar. If a spot has a p-value less than 0.01, the Cy3 and Cy5 labeled transcripts detected by this spot are considered to be deferentially expressed in the two corresponding samples.

Yes, for clustering analysis of multiple datasets, data adjustment includes data filtering, Log2 transformation, and gene centering and normalization. Data filtering will remove clustering values from the data set (detected signals or detected ratios that are below a threshold value) Data centering and normalization will transform Log2 values using the mean and the standard deviation for individual miRNA across all samples

We will perform clustering with a hierarchical method using average linkage and Euclidean distance metric. The clustering data can be visualized using one of the several microarray programs, such as TIGR MeV (Multiple Experimental Viewer) (the Institute for Genomic Research).

Yes, the data package that we send to you contains sufficient raw data and array layout information for you to carry out you own data analysis.

Additional Questions

Currently the array chips are not available for separate purchase without the service. Our technology utilizes a microfluidics chip that requires additional liquid handling equipment not available outside our lab. In the future we hope to be providing this equipment so that users may perform their own experiments.

We have a very well-trained and knowledgeable staff of scientists here at LC Sciences. If there is some specific experimental work you need done, we would be more than happy to put together custom service package designed specifically for your research needs.

Yes, they are listed on our publications page.

Sure, please contact us for publication assistance.

For other questions please send us an email at service@lcsciences.com or call us at 1-888-528-8818