Using RNA-Seq Library for qPCR Confirmation

[Carina1225]

Hi Everyone,

I've tried to so some extensive Googling on this subject but can't find the answer I'm looking for.

I carried out RNA-seq on a bunch of samples on the Illumina HiSeq 2000, and retrieved the cDNA sequencing libraries for each sample. I am done analyzing my RNA-seq data but I would like to confirm the gene expression changes I saw with qPCR. Can anyone please tell me if it is possible to just use the sequencing library as a qPCR template, or would that cause problems (like the adapters getting in the way)?

If this is possible, can someone please direct me towards a protocol if there are any special conversion steps that need to occur before using the cDNA in the qPCR?

Thanks for your help!

Carina

[kerplunk412]

Hi Carina,
It should be possible to perform qPCR on your RNA-Seq libraries. Shorter amplicons would be better, as there is a greater chance the entire amplicon will be present in your library.
That being said, if you have any of your starting material (RNA) left, it would be much better to confirm the changes with traditional RT-QPCR on that material. QPCR on your libraries only provides another measurement of the molecules present in the libraries, while additional RT-QPCR is a step closer to an independent measurement of the RNA molecules in the starting material.

[Carina1225]

kerplunk412,

Okay thank you so much for the information! I still have some RNA left for each of the samples, so I can just use that, I just figured since I already had the cDNA libraries that maybe it might come in handy.

Thanks again!