Hi all! I have come across something weird in my latest qPCR study, and I would like to hear your input. I have exposed fish to a drug and have measured the expression of a couple of genes a a specific tissue. What I have found is that I have a strong trend of decreasing mRNA expression WITHIN my exposure groups, and that this trend coincides with the order in which total RNA was isolated from my samples. The lab assistant doing the RNA isolation did it non-randomized and in groups of six samples. 230/260 and 260/280 ratios were all above 2, gel electrophoresis showed no degradation and the total RNA concentration did not show any discernible trend. Could it be that the within-group trends I'm seeing is due to the fact that it takes a little longer to isolate RNA from the "later" samples (some thawing of tissue etc) and that some mRNA is degraded during this time? And I have to point out that the trends are in the raw expression data, and disappear when I normalize to the housekeeping gene (which also shows the trend).
So what do you think? Have you encountered anything like this before? And most importantly, does this affect the credibility of my data?