I would like to validate miRNA NGS data by qRT-PCR. This theoretically could be done from total RNA samples (the one used for sequencing purpose).What approach should I use to isolate the miRNA or synthesize the miR specific cDNA using total RNA sample.I know there are kits available which directly uses the sample for miNRA isolation and cDNA synthesis.But in this case I can't use them because I have total eluted RNA with me.
Dear Parul...
I guess Exiqon's microRNA qPCR platform is a good system for validating NGS results.
Best Luck
Total RNA does not necessarily imply microRNA fractions were isolated.
Did you use a column that also isolates microRNA?
You can use the NCode miRNA First-Strand cDNA Synthesis Kit + SYBR green primers to amplify many microRNAs provided they are 1) not related to closely-related sequences (i.e. let-7), and that they are mid-abundant in expression. Msg me if interested in details.
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