Hi,
I try to optimise the qPCR of both the ITS regions. Before I made this optimisation by PCR and the 8 points of the standard (from 10 ng/µl diluted 1:5 for 7 times) work very well and I obtained the amplification of all the points. When I tried the qPCR (same mix and thermoprofile) I did not see any amplification until the 35th cycle. At the end of the analysis, I saw the melting curve, the product that I saw in the qPCR was not correct. I tried to run, in the same moment, the same samples for the detection of ITS and D2LSU and for D2LSU worked very well and for the ITS did not work.
I did not understand what happen in the qPCR.
I used the primers ITS1 and ITS4.
If this worked in conventional PCR, real time qPCR it should work again? What is size of amplicon 400-600 bp?? In that case you should try to design primers will lesser fragment size, for RT qPCR it is not advisable to have longer fragments.
Start by choosing just one primer pair at a time to optimize. It's hard to troubleshoot when you have that many variables. Not seeing amplification until late and having the incorrect product means your reaction failed. You either amplified an artifact or primer dimers. First, make sure your primers are reliable in standard PCR, then test them for qPCR. Last, you can set up qPCR with more than one set of primers. Good luck!
@Katie A Clark Thanks but I think that the problem was the mix of the qPCR. When I tried the standard in the PCR the NTC was completely clear withou dimer of primer. If I prepare the samples with the mix of qPCR and ran in the PCR, I obtained the amplification of only two samples. For me the problem are the ihnibition of SYBR or the dUTP.
Thanks so much for your answer.
@Yashwant Chavan in my case the fragment is 500-600 bp. I did not try the conventional PCR but I work with the qPCR and not RT-qPCR. I decided to quantify the full ITS regions because I found more papers that made it.
I think that the problem is the SYBR or the dUTP, because when I prepare the samples with the mix of qPCR and I ran in the PCR, I saw only the amplification of the first two samples.
I hope that I found the problem because is the first time that I see the things in the qPCR.
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