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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