I am validating the expression of several genes by qPCR to confirm transcriptome analysis. After many inconclusive runs, I realised my housekeeping gene (GAPDH) was downregulated in my transcriptome analysis; unfortunately, all genes used as housekeeping genes are downregulated in my 3 transcriptome experiments. I thought I would represent my data using the “Average of Cycle Threshold (ACT). Is this correct? How can I better show my data?
Thank you all
You said that you have transcriptome data, how about genes that didn't show differential expression, ones that are nearly 1.0 fold change in expression. Have a look at their read counts and pick something relatively abundant?
You should have a plan for how to analyze your data BEFORE you collect any data.
Most folks use the "delta delta Ct" method with the Pfafl correction.
You try to validate the differential expression of some genes of "transcriptome data" by qQCR and at the same time use the information from the same data about other ("housekeeping") genes being correct? How can you be sure that the whole transcriptome data is biased? Why do you think that the differential expression of some genes are estimated correctly, but of others it is not?
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