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Act1 mRNA decay is not linear

qPCR act1 mRNA decay

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#1 aznbabie



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Posted 18 September 2014 - 08:05 AM

Hello everyone,


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


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