I am calculating mRNA half lives between two genes that I am interested in. I performed an analysis of the housekeeping gene (Act1) on a Quantity (no of copies) vs Time (min). The graph is not linear (it's polynomial), making the results for my other genes invalid.
Though the other two genes that I was looking at had "regular" looking decays.
What kind of troubleshooting should I do?
My general protocol:
-Preformed RNA Polymerase shutoff with thiolutin (3ug/mL) over 60 mins (8 timepoints)
-Extracted RNA with a kit
-Performed cDNA first strand synthesis and cDNA cleanup with a kit
-qPCR different dilutions of a pooled sample of all timepoints for "actual" standard on the actual experimental plate
-Found the best dilution (1:5), performed cleanup of qPCR 1:5 dilutions
-Followed qPCR map and performed 1:5 dilution for all timepoint samples and ran plate