Hi,
I am running standard dilutions (1:5) of some cDNA template to check the efficiency of primers. I found to have apparently PCR inhibition, as the efficiencies that I am getting for all the targets are over 110 % (all are around 130-140 %), with R2 >0.98. However, I can't understand where I can be getting PCR inhibitors.
I measured the purity ratios of the RNA after isolation, and they are 260/280=2.09 , 260/230=2.23. I also measured the purity of the cDNA after the reverse transcription , and the values are 260/280=1.77 , 260/230=2.22. Although the 260/280 ratio drops ( I guess because of the reverse transcriptase), I don't think is too dramatic.
I even dilute the template for the first dilution point, to put no more than 50 ng per reaction. If I am introducing inhibitors I think it can only be during the real time qPCR. I am using the LightCycler 480 SYBR Green I Master kit from Roche, but it is brand new.
I analyzed the data with the ThermoFisher Cloud, and I am getting Amp scores over 2 for all reactions, so the quality of amplification is good. I only see one peak of melting curve for each reaction. For all these reasons I don't understand how I have PCR inhibition.
Can it be an issue of the set up? I thought that working out of optimal temperatures and cycle times will decrease the efficiency below 90%, but not increase it over 110%.
Does someone has any clue of what can be happening?
*From my little experience with qPCR*
How do you extract your RNA for cDNA synthesis? Do you use Trizol reagent?
I monitored my last run of samples from RNA extraction to cDNA synthesis and real time qPCR. What i realised was that one of the RNA samples was contaminated with a little trizol reagent eventhough I got A260/280= 1.98 and after DNase I treatment A260/280= 1.98 meaning my RNA is pure and good for the PCR.
The qPCR reaction however had inhibition.
I suspected the little trizol contamination to be the source of the inhibition
I hope this helps
Hi Nathaniel, thanks, but in tis case I used RNEasy columns. Did you check the A260/230? It indicates the presence of other compounds that can contaminate and inhibit the reactions, but in my case the values are high, that's why I don't understand what is happening.
You may not have evaporated all the ethanol out from your extraction, especially when using a Trizol/chloroform extraction. Ethanol contamination can inhibit PCR.
DNA purity ratios do not always make sense. I would try to dilute my sample at least 1:3 prior to running qPCR. If there is an inhibitor, it is in your sample.
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