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I have been having an apparent problem with gDNA contamination in my no-reverse transcriptase (NRT) controls by the appearance of fluorescence peaks in my qPCR data.

I have/am trying multiple DNase treatments (Turbo DNase, Life Technologies) and RNA-clean up (Qiagen, RNeasy). The limiting factor I am worried about is that I am going to destroy all my available mRNA with multiple treatments/clean-ups.

My question is - can I lower the number of cycles I use to measure fluorescence? Is there an aspect that will go awry (in terms of the experiment itself to data analysis) if, let's say, I lower the number of cycles to 25-30? All of my contaminations start to appear within the range of roughly 30 cycles and above (as determined by the Ct values).

Please let me know if you need any other information that could help for an answer!

Best

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  • $\begingroup$ Are your targets getting into the exponential phase above background before your contaminants are? $\endgroup$ – CKM May 16 '18 at 1:44

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