Most RNA-seq analysis methods lead to log2FoldChange, whereas in qPCR, analyzed via ΔΔCT method, also gives a foldchange result. So i wonder, in ideal conditions, should those 2 foldchange results obtained from different experiments be roughly the same? Or at least, agree with each other? If there is always discrepancies between RNA-seq and qPCR results, in normal cases, how big will it be?
My situation is that, the foldchange of my samples from qPCR is above 10, while the one of the same sample from RNA-seq is only 1.3 (p
Ideally yes the RNAseq and PCR results are in agreement. However, there is often some variation. As you say, you are seeing the same trend of increased expression for your gene of interest. It is possible that the read coverage for your gene is low, or if there are other similar genes, reads may be mapped to homologs. Are you using paired tests of the same sample? There could also be sample to sample variability.
Hi Xingyu,
ideally RNA-seq and qPCR give you a very similar answer. You can try and double-check a couple of things: Is the qPCR optimized (product does not come up too early nor too late), are your reference genes actually stable in the conditions you test (you might want to consult the RNA-seq results), are the genes generally of low abundance (which corresponds to higher variability)?
If you still have the RNA sample you used for RNA-seq you could perform qPCR on the exact same samples.
Cheers,
Lukas
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