4 weeks ago by
Spain. Universidad de Córdoba
Wondering again why we keep thinking that validation with qPCR is required for our RNA-Seq experiments. I think time of changes have to come in the sense that we should consider even the opposite, that is, that we should use RNA-Seq information to validate our qPCR experiments
RNA-Seq relay in the single fact that a sequence read, as long as 100 or 150 bases is undoubtedly mapped to our genome or transcriptome. If error arises from sequencing, this mapping does not take place. We can always take into account the quality of mapping and the flag values to filter our mapped reads to get rid of soft and hard clipping that could be discarded. In fact, this filtering should be imperative in my opinion.
In any case, it is certain that RNA-Seq is not a fully bulletproof method. But... If we compare the many different issues that arise from a qPCR experiment such as is the RNA degraded along the experiment?, is there any other complementary RNA hybridizing with mine that interferes with the amplification ?, how well the primers align ?, is there any GC or bias base composition that interferes with amplification ?. Are my reagents and enzyme in good condition?, is my device clean enough to accurately do the measurements ?. Is my assembled genome accurate enough?. These are some examples that in my opinion demonstrate that much many issues can affect qPCR