Is qPCR actually required for RNA-Seq validation ?

Question

[Antonio R. Franco]

I fully understand that a qPCR is required in a microarray experiment, because of many reasons. You are playing with hybridizations and this can lead you to run in many kinds of problems (cross-hybridization, background, false hybridization, etc)

But to me, RNA-Seq is quite different. You are using actual sequences that are mapped to your references. You can also control reads that are not mapped uniquely. And this means actual data that cannot appear as an artifact, thus making me think that maybe qPCR is not required.

In addition, qPCR is not prone to be a perfect technique. However, a lot of papers show validation of RNA-Seq data using qPCR, and I am wondering which is the reason for that. I understand that it depends on several facts, like when you are mapping to cds only (where you use to run the qPCR) or to 3' UTR as well that can lead to a different result since 3' UTR expression can be quite variable sometimes..

Do you think that RNA-Seq is actually required to validate RNA-Seq data?. What are your thoughts ?

[microfuge]

This discussion on twitter is quite relevant in this regard.

[John]

Amen. PCR is just read mapping with enzymes in a tube.

Hahah, these guys :'-D

• What a cell does use to find foreign DNA - 22bp miRNA primers
• What the cell doesn't use to find foreign DNA - reference genomes for all bacterial species from which they can align suspicious DNA to.

[Farbod]

I think it is critical question that most journal reviewers do not like to ask it from themselves.

We have used RNA-seq for trapping a gene/transcript with low expression that was biased between sexes. our aims was this that the farmers with low income becom able to use normal (cheaper) PCR to determine the sex of their animals before a certain age (that secondary sexual characteristics will develop). We aim a gene that was present in one sex and absent in another and we have used RNA-seq because there was no reference genome for our species of interest. We have represent this fact in many parts of our paper (abstract, background, discussion and . . . )

Interestingly when we proceed to publish our paper, the reviewer asked for the qPCR results! which was totally valueless in our design!

So, in my idea; according to the research design, it could be important or useless.

[John]

I would say it is, for 3 reasons:

1. You are unlikely to be able to publish without it, so it's a very real obstacle to your success.
2. It's a much simpler protocol with far less steps that introduce a bias in some way or another. Tried and tested to be accurate.
3. It's quantitative, as the 'q' in qPCR suggests. Some RNA-Seqs claim to be quantitative with spike-in controls, but i'm still personally skeptical.

Answer 1

[Devon Ryan]

It depends on what you want to do with the results. qPCR is just a cheap and easy orthogonal technique, the kind of thing you'd do to confirm any experimental results. If you plan on doing different follow-up experiments given the results anyway then there's no need to do the qPCR (if a reviewer asks for it, then point to the other experiments and say, "to what end?"). If the DE results are the main point of your story, then they need to be backed by an orthogonal technique.

[Farbod]

Dear Devon hi,

That "to what end?" was very nice!

~ Best