We can screen human, mouse and rat samples for micro RNAs by real-time qPCR using Exiqon reagents and arrays. As with any screening protocol, it is critical that total RNA samples be prepared to include the smaller micro RNAs. Purification of miRNAs is not required but the proper kit should be used. Sample quality is always important but more so for this kind of screen. As with microarrays, an Agilent 2100 chip will be run with all samples to ensure RNA quality. Samples that fail this step will be returned to the investigator. Samples should be at 20-100 ngµl in nuclease-free H2O. Unlike expression analysis of individual transcripts, a single cDNA reaction is performed using ≈ 20 ng of total RNA. The entire cDNA reaction will be added to a single large PCR master mix volume and dispensed over 2 384-well plates bearing miRNA-specific primers (Exiqon) using liquid handling robotics. Six control small ncRNAs, a spike-in and inter-plate calibrators are included for each plate array.
miRNA hits identified by microarray or real-time qPCR has to be validated using a much larger sample set by real-time qPCR. Sample preparation is the same as for an miRNA screen above. The Agilent 2100 chip not only shows RNA quality but also the presence of miRNAs within the total RNA sample. As with the miRNA screen described above. A single cDNA reaction is run for each sample. The cDNA is then added to PCR master mix and dispensed in replicate wells over a 384-well plate followed by miRNA-specific primers. Samples should be provided at 25 ng/µl in nuclease-free H2O. Validation sample sizes are set at 16, 32 or 48 samples for 12, 6 and 4 primers per 384-well plate, respectively.
Data will be provided in an Excel workbook for each miRNA. Data will be analyzed by the director and supplied to the investigator in an Excel workbook. Analysis beyond that done by the core director will be the responsibility of the investigator.