I'm having some trouble with my RT-qPCR. Some of my amplification curves (delta RN vs cycle #) look as expected, but others have a distorted curve. High starting background that drops, followed by a short linear phase, and a drooping plateau. I've attached a picture, where the green is the expected curve and the blue is the questionable one.
The samples were done in technical triplicates and all curves look about the same. The dissociation curve also looks fine with a single sharp peak.
The ABI software is still able to determine a Ct value. My issue is that I am trying to import the software into the program LinRegPCR (linregpcr.nl) to more accurately determine the primers' PCR efficiency. Doing so, the program is unable to determine the linear phase of the curve for the distorted plot (second attached graph).
What went wrong? Why does the amplification plot of this sample look so odd? Is there an issue due the discrepancy in amplicon expression between the two samples? Could the high expression in the "distorted" sample be the cause of an usual graph?
Thanks for any help!
linregpcr.nl
Dear Sam,
I did´nt understand the question at all. But my tip is dilute and dilute and dilute the sample . Is the sample denaturated? Thawing and freezing many times or shaking after isolation? This may be a problem....
Nice greetings from lower austria
Heidelinde Schranz
Hello Sam,
I'm not familiar with the LinReg software, but in order for you to assess your curves, you may need to view them using the linear scale, the Rn not the delta Rn https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/pcr-understanding-ct-application-note.html
This should help you get rid of the noise background and view your curves in a more comprehendible fashion. On a side note, when you have a inconsistent plateau it means something inhibited the reaction during the process, however looking into your curves this doesn't seem to be the problem. I think they are perfectly fine, your first sample is very high in CT value, and plateaued fairly fine until it reached the end of reagents. try viewing the run in a Rn fashion and let us know if you still feel your reactions are not functioning fine.
Hi Maha -- Thank you for the advice! Viewing as Rn vs Cycle #, the plots (I think) look fine. Though it's clear that one sample is much more highly abundant than the other. Perhaps this is problematic in my particular software.
I'm not sure what you mean when saying highly abundant, I would say your first sample has a much higher amplicons compared to the later, your pcr reaction cycles terminated at cycle 40, had you ran it a little longer it would have reached a higher peak prior to plateau. The reaction terminated while the sample is still in the log phase, hence I think this is a perfect run. if you dilute your first sample 1e-6 or 10e-7 and run the reaction for 45 cycles instead of only 40, they will both pleateau in perfect alignment.
Hi Maha -- this is wonderful advice and my next step. Thanks for your help!
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