Hi! I've been running qPCR on some drinking water samples with a primer/ probe FAM assay. In any given run, there have been at least a few samples with artificially high fluorescence reads, sometimes even higher then 10^9. The curves associated with these sample wells are flat, and when the qpcr products are run on an agarose gel, the target band is not present or is not nearly as bright as the band of a midrange standard. I was wondering if anyone might have any thoughts on what's going on.
The DNA for the samples was extracted with phenol-chloroform-isoamyl, the samples are diluted 1:10 before being used in qPCR, R^2 is >0.99, and the efficiency is ~80%.
Thank you!
Hi Sophia,
As what you have described, I would believe this is not a true amplification. you may probably check at what cycle the fluorescence arise, if these arise before cycle 15, then it is more likely this is due to false signal, that means there is no amplification in the samples.
If your assay is associated with ROX passive reference dye, you may check the ROX curve as well, this will probably give you some ideas if anything went wrong with the reaction.
The most possible cause of this issue that gives the false signal could be the template too high, and this can be frequently seen in Ct value below cycle 15.
You may further validate this by quantitate the sample concentration.
Hope it helps.
Hi Sophia
There might have been some noises at the beginning of your amplification curve which might go a bit above the threshold line. In this case even without a good amplification you have a very high copy number. To have better results, you may set your threshold manually to avoid noisy samples interrupting your results.
Good luck
Dear Sophia Blanc please show the graphs and gel, it could help us better understand your case.
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