no increase in fluorescence in my real time PCR - (Feb/02/2011 )
I have a trouble in my real time pcr runs. The gene that I have interested in does not work in real-time pcr. I have optimized the conditions for the primer in PCR before, I have tried both genomic DNA and cDNA as template and observed nice bands on the gel . However, when I have switched to qPCR I couldn't observe any increase in the fluorescence (using cDNA as template). Also I have loaded qPCR products onto gel and as expected I could not observe any bands.
I am using FastStart SYBR Green Master from Roche and the machine is Rotor-Gene 6000. While my reference gene works very well in qPCR runs , my gene does not. I have tried different dilutions ; first I've tried 1/20 & 1/200 dilutions. Then thinking that the template might be too dilute I've used more concentrated cDNAs (no dilution & 1/5 ).
Icould not find an explanation for this situation. Do you have any suggestions or possible explanations?
Thanks a lot:)
The fact that you have an assay that works just fine (your reference gene) suggests to me that everything is working just fine. Your main problem is the primers that detect your gene of interest. My main recommendation to get this to work is to design at least two, is possible three, different assays to detect your gene of interest. In my experience even the best primer design tools sometimes fail to create a good enough assay and things simply do not work. When you test at least 2 or 3 assays more often than not at least one will end up working very well.
Thanks a lot for your suggestion.I want to mention that my primers are working just fine with RT-PCR and they are also appopriate for qPCR, product size is 132 bp (not so big). Therefore, I think my primers are ok.
I suggest lowering annealing temperatures in your qPCR compared to your conventional RT-PCR. Different PCR kits may contain different salt concentrations, which influence annealing temperatures. You could also try a RT-PCR using your SYBR-mix in your conventional PCR machine.