Does anyone have experiences with nested duplex RT-PCR? We´re trying to develop screening PCR based on RNA templates including internal genomic control. After RNA extraction, we reverse-transcribe all the RNA into the cDNA. After that, we´got 1st Round with primers only for gene of interest. The 2nd Round includes both, primers for gene of interest and either primers for internal genomic control. The visibility on the ELFO is good for our target gene, but the visibility (or sensitivity) of positive internal control is too low, however the number of copies (of internal positive control) was 100,000 in RNA extraction. The template for the second round is the 1st PCR product 50x diluted.
In duplex PCR, the amplification of fixed copy number internal control depend on amplification of other target. It is usual that if your target amplify early (and strongly), the internal control may fail - due to exhaustion of reagents and inhibition on polymerase. Usually internal controls are used to validate negative sample data - to ensure that you got a negative from true negative sample, but not due to any aberration in PCR reaction/extraction.
Hy Robert
According to my experience do not exeed first amplification round, 15 cycles is enough, may be 10 already sufficient. Try another Polymerase-mix like Quantifast from Qiagen or Sensifast from Bioline.
I wish you success
René
Hi Róbert,
A couple of things could be going on here. First off it's possible that the primers unfortunately bound to some unexpected bit of DNA, which means of course that the internal primers would not bind to those PCR products during the second round of amplification. Another possibility, as M N Manoj pointed out, is your gene-of-interest primers could be outcompeting the internal primers.
Hope this helps!
Lance
Hi Róbert,
I am not sure if I understand your approach correctly and I have some questions. You have isolated RNA, reverse transcribed it into cDNA (how much RNA in which volume?) and want to detect an housekeeping gene transcript and an target gene transcript? Which priming strategy did you use for cDNA synthesis? Using the cDNA (which volume?) you started a target gene specific PCR and used 1µl of the 1:50 diluted PCR reaction for the second PCR. This PCR reaction contains both target and HKG primer pairs. And after this PCR you detect the target gene only? How did you quantify the 100000 copies within the RNA (in the total volume, or the amount you put into the PCR)?
Regards
Norman
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