I am new to RNA-seq analysis and I wanted to ask about comparing expression in different genes. I apologise in advance that this question is quite basic. I used Cuffdiff to perform a differential expression analysis and validated several candidate genes identified using in situ hybridisation. While the validation of most genes confirmed the expected differential expression, it was difficult to decide on the cut-off for when no expression could be expected. For instance gene A had a FPKM of 1000 in cell1 and FPKM of 90 in cell2, validation showed expression in cell1 but not cell2. However, gene B had a FPKM of 80 in cell1 and FPKM of 3 in cell2, and validation showed expression in both cell1 and 2, though it was stronger in cell1.
Since FPKM is normalised for gene length, I assumed that the FPKM of different genes should be comparable. Am I wrong in thinking this? And could there be any reason that validation using in situ hybridisation of some genes show no expression when there are transcripts according to the transcriptomic data other than the sensitivity of the probe in detecting the gene?
Thanks so much!