I have performed a reverse transcription using the High Capacity cDNA RT Kit but due to an error in programming the thermocycler instead of one cycle (25C-10min, 37C-120min, 85C-5min) the last step continue for 29 mins, so the cDNA samples have been at high temperatures (85C) during half an hour aprox. I want to do rt-PCRs with those samples, do you think I can use them? I will be doing a test with some samples anyway but I would like to know what can be the consequences to have cDNA at high temperatures.
Should be just fine. Remember: this is DNA not RNA, now. DNA is much, much more thermostable. Once you've made it (the "37 degrees for two hours" step), it's made.
The very qPCR experiments you are planning to run will, after all, feature many, many cycles of 95 degree template melting, and nobody expects DNA to degrade during thermal cycling.
RNA tends to degrade fairly rapidly above 65 degrees, but DNA should be just fine.
Dear WA Gadalla , yes your cDNA have to be ok, but - it is only my opinion - do not use it for important experiments. You may receive interesting results and ask yourself any time - may be the results affected by cDNA synthesis. I have felt it ). Indeed, in the case of problems with reproducibility of your experiment you may have troubles and extra time at +85 can disturb your mind. Good luck.
At that point, the cDNA had already been synthesized. I don't think it's degraded. I suggest you to do an end-point PCR and check that amplify without a problem.
It should not be a problem, cDNA is thermostable. I would suggest you try them once by PCR. If you want, you can just repeat reverse transcription for one of your samples and have it as a kind of control along with the rest of the samples.
Good luck
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