I ran tophat-cufflink-cuffdiff of my RNA-seq, and got the cuffdiff "gene differential expression" file. I noticed that for some genes, they appear multiple times, and each time there will be different value in my siNTC vs. siGene, sometime higher in siNTC, sometime higher the other. For each gene, isn't it supposed to only show once with values in each experimental condition? And I have been trying to validate some targets from the analysis, but had no luck. The targets I picked have over 5 times expression difference according to the analysis, but my RT-PCR just show very little effect, using the same RNA that was run for Seq. For the gene that I K/D using siRNA, it shows about 95% knockdown efficiency, and about 90% from the RT-PCR, so that is normal. I am really confused with the analysis, I also tried the HISAT2, but same thing. Could anyone help me on this? Thanks a lot!!