I would like to validate miRNA NGS data by qRT-PCR. This theoretically could be done from total RNA, isolated small RNA (since that was used for sequencing library preparation) or the sequencing library itself. Which one would you recommend and why? My feeling is that because total RNA and the small RNA fraction undergone some processing steps, they would not be the same for validation as the library. Furthermore, what would be the best for normalising the qRT-PCR? Spiking for example? Any comments are appreciated.
Hi,
People usually do not use sequencing library itself as a template for qRT-PCR. You can use either total RNA or small RNA fraction.
I use U6 snRNA for normalization, but any other structural RNAs (rRNA, snRNA, snoRNA, etc) would work for this purpose.
Perhaps another option is sequencing independent total RNA isolations for the same samples, ie replicates. This is more expensive though.
I agree with Dooyoung, you can use the small RNA fraction. U6 is used frequently however I've found that ribosomal RNA 5S is best (I tested it in maize).
Ypu can't use the sequencing library because whatever biases were generated there will be reflected in your qPCR results. I would do it on total RNA.
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