We can screen both human (miRBase 10) and mouse (miRBase 13) samples for micro RNAs by real-time qPCR. As with any screening protocol, it is critical that total RNA samples be prepared to include the small 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 200 – 250 ngµl in nuclease-free H2O. Unlike expression analysis, a single cDNA reaction is performed using 0.5 – 1.0 µg of total RNA. The entire cDNA reaction will be added to a large PCR master mix and dispensed over 2 384-well plates bearing miRNA-specific primers (Qiagen) using liquid handling robotics. Six non-coding RNAs are run for each sample for data normalization.

miRNA validation: miRNA hits identified by microarray analysis or real-time qPCR can 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 mixes bearing miRNA-specific primer. Samples should be provided at 250 ng/µl in nuclease-free H2O.

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.