normalization to “housekeeping” genes of RNAseq data vs qPCR result

normalization to “housekeeping” genes of RNAseq data vs qPCR result


It is relatively common to use “housekeeping” genes when performing qPCR experiments. I would like to know how common (or uncommon) that a gene expression found in RNAseq can be replicated with qPCR.

Indeed, I usually get fairly comparable results between these two experimental techniques. However, I shift to study metabolism-related pathways recently and found that many of the transcriptional changes are not consistent between RNAseq and qPCR. One possibility is the metabolism shift will induce a large array of gene changes, that might also include the “housekeeping” gene I chose (I usually use actin or 36b4).

an example is: we found that from RNAseq, the actin level is downregulated after the treatment, thus, indeed make using actin as a “housekeeping” gene invalid. On the other hand, I wonder if that down-regulation of actin could result from generally up-regulation of all other genes? Given that many RNAseq will still be normalized to the total library size, thus, I am not sure the analysis pipeline, such as DESeq2, can deal with this.

I wonder are there any means to correct this, or at least diagnosis on the data structure/ quality so that I can know I should interpret the RNAseq/ qPCR result with cautions?





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