General Q: In case RNA-seq and qPCR don't agree, why is qPCR supposed to be better ?

I have never seen anyone asked to do this, and wouldn't ask for this if I were reviewing a manuscript unless there was an excessive amount of PCR during library preparation or something else wrong with the methodology. Sorry Karthik, I think you were unlucky with your reviewer :-(

You should definitely have biological replicates though.

I think yes! it is always necessary to validate functionally the selected potential genes obtained from RNA seq data for the given trait. RNA seq gives many genes showing differential gene expression, which could be due to some other factors as well but to narrow down for the exact candidate genes we should always validate the genes through qRT-PCR.

Thanks all. Yes, we do have biological triplicates that is why I had consider not to do qPCR to validate the DEG.

Depends on who the reviewer is! But usually I'd say no.

Depends also on how much your follow-up research relies on this gene(s) of interest being differentially expressed. If very important, then do the qPCR. If you're more interested in overall patterns (GO analyses, how many genes diff. expressed), then don't do it.

Also, if you have other types of data backing-up why you expect this gene(s) to be diff. expressed, you can probably argue with the reviewer that the qPCR is not necessary.

General Q: In case RNA-seq and qPCR don't agree, why is qPCR supposed to be better ?

I think that we will use qPCR from the RNA we have extracted just to check some known genes which regulated expression we know, just to compare how similar are both assays.

Thanks all.

Wouldn't it depend on the conclusions being drawn vs. the depth of sequencing? For example, if I am reporting new transcript variants using a lesser than 100million-read-data from a human tissue, it would be good to validate them by RT-PCR followed by sequencing (at least important new variants being discussed); if I am concluding differential expression of certain known transcript-variants with less than 50 million reads, it would be better to perform qRT-PCR of important transcripts at least.

Related to Kshitish K Acharya's post on splice variants detected by RNA-seq, I refer to Jan Helleman's blog post on confirmation of RNA-seq splice variants by RT-qPCR. Results will be published in Nature Biotechnology soon, as part of the SEQC study.

http://www.biogazelle.com/novel-splice-junctions-identified-rna-sequencing-studies-noise-or-interesting-biology