PCR - qPCR inconsistent - To explain differences between normal PCR and qPCR (Jan/15/2007 )
I try to evaluate a gene from endothelial cells, I use a previously reported primer pair that works well in normal PCR. But, when I do the qPCR using Sybr green, I don't see signal, neither amplicon in melt curve. The Sybr mix works well then I get good signal with GAPDH with the same template. I use the DyNAmo from Finnzymes and a Perkin Elmer 5700 cycler. Can anyone help me?
how about your PCR amplicons' size?
Amplicon is 201 bp. I have used from 1000 to 200 nM of primers and a lot of cDNA, but there are not specific signal.
I use a previously reported primer pair that works well in normal PCR--------
Do you mean that you do the normal pcr yourself or you just know it from the paper? if you just get the primer sequences from the paper, you should test themyourself. if the primers really work well, then you should know the follow,
1, are you sure that the target gene express in endothelial cells? Does it express in a very low level ? how about the quality of your RNA?
Do you have a postive control?
2, Did you run a agarose gel of your sybr green pcr products?
3, Did you use the same amplify condition in the sybr pcr and nomal pcr?
just my own suggestion, maybe it is no use.
I use the reported primers in our samples of endothelial cells in normal PCR and it works well. I get the correct size amplicon using endothelial cells (which is the positive control). I do the RNA extraction with Trizol, and normally it works in other experiments, even GAPDH setted up simultaneously.
I tried two step and three step protocol in qPCR. I heard that Tm of primers pairs must be similar or less of 4 degrees, my primers have exactly 4 deg difference. I am trying modify uneven concentration of each primer, but I have no result yet.
I try to detect two isoforms of transcript, and the large isoform amplify well. The short isoform don't work, and we cannot change de primer design, because, this is the only amplicon to differentiate each one.
since the primers worked well in your normal PCR reaction. ( just one band in the agarose gel ???) i think it will also work in the sybr pcr. just use the same amplify condition in the sybr pcr. if you use your homemade taq, set up 2 reactions, a normal pcr and one with the adding of sybr in the normal system. then run a agarose gel. you can know is there any problem with your reagent .
I tried changing primers concentrations and raising the template amount. Efectively, I found a "pair of concentrations" that works well and I get finally an amplicon detected by their correct Tm. I appreciate your help, thanks,