I've seen in some articles that real time pcr is more sensitive than the conventional one, so I don't know if I should repeat the process using the rt-PCR to ensure that the gene of interest isn't really present.
If you used proper controls (H2O, negative, positive) when you genotyped you should be okay.
If your reagents are ok and primers are fine you should get the results with conventional PCR too and there should not be any difference if you repeat it with rt PCR.
Hi, I think it could depend on the enzyme you are using. In our lab we use a specific commercial enzyme master mix for RT-PCR, and it works very well, well-optimised with a high polymerisation rate, etc., much better than the Taq we would put in a conventional PCR, so I would say it is quite possible you could get better results with real time than conventional PCR. Another factor is running a gel vs detecting fluorescence, which may be more sensitive to small amounts of product.
Either way, I would be careful of concluding that "the gene of interest isn't present". Just not at detectable levels...
Sensitivity in this context is referring to how/when the PCR product is detected as a positive result. Conventional PCR amplicons normally range from 200bp+ and need to be visualized on a gel to confirm positive/negative results. In qPCR the amplicon size is normally shorter (50-150bp) and as the fluorescent signal is released per successful PCR, it measures target per copy and is independent of whether we can visualize it on a gel or not.
If you are using the same primers for PCR and qPCR, theoretically it should give you the same result so long as your PCR assay designs are reliable. The cycle number used is quite important as well as how much target you expect in your starting samples (if you expect a lot, 25-35 cycles is norm while target in low concentrations may require 40-45 cycles). If you want to continue using conventional PCR, increase your cycle number by 5-10 and be sure to include a negative control. Run your PCR products on a gel with a ladder, a positive result is if your sample exclusively has a band at the expected amplicon size.
results must be same in both, if you are sure with conventional PCR (getting expected band size), then no need to again do Real time PCR, instead you can confirm by sequencing.
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