Should I validate my RNA-Seq results by qPCR?

As we know microarray deals with probes.

One problem is that in a single experiment the limited number of hybridization probes can hardly represent the whole transcript population in the sample. This would result in a probe bias and reduced linear range. So the standard procedure is to technically validate microarray results by qPCR. However, as RNA-seq detects the entire transcripts and through a series of quality control and normalization the technical bias is greatly reduced. On the other hand, qPCR has its own prob bias. Personally I have not seen many people include qPCR in their RNA-seq paper. But for those who did qPCR validation, they found good correlation between the two approaches.

correlation-rnaseq-qpcr

Weather qCPR is really necessary for RNA-Seq, here are people’s opinions:

We had considered performing qPCR studies to ‘re-validate’ some of our gene-expression findings but there is little evidence that qPCR analyses from the same samples will add any extra utility to our data so we decided to eschew those experiments. – Dave Bridges

In my opinion, the validation by qPCR was not essential, but is useful. – a paper reviewer.

Validating your experiment is not that necessary I believe; precising an observation is nice on the other hand. Usually, I see articles comparing micro-array and RNASeq data, not RNA Seq and RT-qPCR. – a Biostar thread

Although validation is required by a lot of journals for publication, it is still debatable whether qRT-PCR validation of differentially expressed genes is still necessary…and how to do it exactly if the answer is yes. – Design and validation issues in RNA-seq experiments

In summary qPCR could be more helpful if no biological replicates were used in RNA-Seq. However, if the reviewer or the journal requires such validation even if replicates are indicated, it’s out of this discussion and you just need to do it.

Z. Lu avatar
Z. Lu
Data scientist, bioinformatician, retro fan and web lover.
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