Forum:RNAseq validation using qPCR

Question

[phalange.re]

Hi guys,

I've run qPCR validation of my RNAseq data to address the comments of one of the reviewers of my manuscript. I'm just a bit confused on how to compare the data from RNAseq and qPCR. Comparing the fold changes (as seen in many papers) doesn't seem right to me. I've used edgeR for my RNAseq DGE analysis and 2^-DeltaCq (using geo mean of three reference genes) for the qPCR data. These data don't seem comparable based on how different these analyses are. Could you please let me know how do you guys usually compare these two methods? Is fold change comparison really OK?

Thanks a lot!

Answer 1

[Sam]

The way I do it is to correlate the RNA Seq count with the Delta CT values (or the Delta CQ in your case?). If you have a high negative correlation, it means that your RNA Seq count is more or less representative of the actual mRNA concentration. Also, if you are interested in seeing if the fold change, or more precisely, the significance of difference observed in RNA Seq can be validated by the rtPCR, then you can follow instruction of this paper.