hi , if you please i need to know what are the reasons of the false positive results in real time RT-PCR of non specific template reaction, the primers are not similar to the non specific template while the probe is close similar to the non specific template? could i get an explanation ?
Definitely, my problem is in the reactions that have a DNA but it is supposed to be non-specific for the primers and probe. After checking the primers and probe sequence, I found that the primers were completely non-specific for this DNA but the probe was specific partially. so, my question here is, can the reaction be falsely positive because of the probe?
thank you
Unfortunately, a common reason for amplification in the no template (or no RT) controls is contamination. Either there was residual DNA carryover during cDNA synthesis, or a reagent was contaminated with residual template (pipetting mistakes, or aerosols, or using dirty pipets, or not using filter tips). RT-PCR is very sensitive so it doesn't take much template to get a signal.
thanks indeed for your interest and answer Amy Klocko ,
I think it is not the reason for my case as the non-template negative control is always clear, my problem is in the reactions that have a DNA but it is supposed to be non-specific for the primers and probe. After checking the primers and probe sequence, I found that the primers were completely non-specific for this DNA but the probe was specific partially. so, my question here is, can the reaction be falsely positive because of the probe?
Do you mean that you blast the primers and probe sequences against the non specific target and you found a degree of matching between your probe and the non specific target?
- Badges
- Science method