Hi everyone,
To those who are studying gene differential expression by using RNA-seq, can you share with me the protocol you follow for validation of candidates genes by RT-qPCR. I mean the PCR experimental design and protocol?
I'm working with bacteria and right now waiting for the RNA-Seq sequences to get our of the Illumina machine. So after analysis I need to validate the results and as it is the first time for me to deal with qPCR it would be great if you can give me some advice.
Thank you in advance!!
The important thing to do is to have several internal controls rather than just one. So in addition to a specific primer pair that amplifies each of your candidate genes of interest that show differential representation in the RNA-seq population, you need to select a few other genes expected to be house-keeping genes and expressed similarly under the control and test condition. Most people use just one internal standard but that is too risky. The problem is that no gene is totally neutral, so if you have several controls, that's better. as long as you follow standard qPCR protocols and avoid saturating the amplification. Finally, it is important to do biological replicas, so
1) you should have an independent RNA-seq result with totally new samples and reproduce the same result,
2) then you move on to qPCR and test each of your favourite candidates with at least 3 internal standards, but you should have 3 biological replicates of control and test condition, that means not 3 qPCRs on the same RNA prep (technical replicas), but 3 different bacteria populations (control and test condition) and independent RNA preps from each, and thus 1 qPCR on each. Much more work to do biological replicas, but absolutely worthwhile doing.
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