RNA-seq already gives us quantitative expression data, so do we really need qPCR validation—or should we be focusing on protein-level confirmation instead? And if RNA-seq and qPCR don’t agree, which result would you trust?
This serves two key purposes. First, it addresses the concerns of reviewers and journals, ensuring the credibility and reproducibility of results. Second, it is particularly important in cases where sequencing data may provide misleading or inaccurate predictions. In such situations, validating whether a gene or its fragments truly exist is essential. This step is critical for non-model species, where genomic histories and reference information remain incomplete or poorly characterized.
Refer this Article Do results obtained with RNA-sequencing require independent ...
I personally think qPCR validation brings little values to the RNA-Seq data, as you usually found more than 100 DEGs. No one would do qPCR to quantify these genes as it defeats the purpose of RNA-Seq in the first place.
I agree with focusing on protein-level confirmation, aka, post-translational evidences.
In our previous research, we found our treatment had led to different expression of multiple cuticle genes and amylase gene in a shrimp. Therefore, we validated the DEGs by microscopy to check on the cuticle integrity, and also quantify the amylase activity and glycogen storage. These post-translational evidences are definitely way more concrete and reliable than qPCR validation.
Article Hsp70 Knockdown in the Brine Shrimp Artemia franciscana: Imp...
RNA-seq is good at broad category creation, but not trustworthy for individual transcripts.
qPCR is much harder to do properly, but is much higher quality and much more reliable than RNA-seq. If you care about the particular expression of that gene in that context/tissue type/environment/treatment/etc.
The general project flow is: RNAseq to get the main ideas/look for "hmm, that's interesting results"; more specific methods to look in detail (qPCR, protein expression, etc.).
Good luck!
Sidney T. Sithole , qPCR used to be the go-to validation for RNA-seq, mostly to satisfy reviewers, but the field is moving beyond that now especially with high-quality RNA-seq data. If your RNA-seq is well-controlled, qPCR isn't always necessary. Protein-level validation is often more meaningful anyway, since mRNA ≠ protein. If RNA-seq and qPCR don’t match, I’d usually trust RNA-seq (assuming good QC).
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