Hi everyone,
I would like to do correlation analysis qPCR (2^(-delta delta Cq)) vs RNA-seq data in terms of fold change. In my qPCR, I have two conditions and one housekeeping genes to be compared. But, how do I represent my transcriptome results (Cuffdiff) since the data does not normalize to housekeeping genes?
Thank you so much.
the log(RPKM) or log(FPKM) values should linearily correlate with the dCt values.
Provided your RNA-Seq data are normalized for sequencing depth, which is covered in both RPKM and FPKM - as Jochen referred to above - you should be able to correlate the expression values of the same gene between qPCR and RNA-Seq after log-transformation. Therefore, such transcriptomics data are normalized to the initial amount of cDNA, which exerts the same effect as normalizing for housekeeping genes in qPCR albeit less accurately.
if you re not using the same biological sample there is a first bias
The library construction and the cDNA from reverse transcriptase are also completely different bpilogical procedure to obtain cDNA or to count reads!
in additon of these main difference at the biological level you have also difference in statistic and in mathematic or the calculation of RTqPCR data and RNAseq.
you the comparison you hsold made shoud be as cautious as if you were comparing data from 2 papers form two different authors
One important thing to take into account is whether the housekeeping gene used in RTqPCR is really not responding to the treatment. You should check this. With only one reference gene you run the risk of getting biased results. With good reference genes and enough replicates, we have obtained high R2 and slopes very close to one and intercept near zero, when using the same biological samples for both RNAseq and RTqPCR,
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