No signal or amplification can result from a number of causes ranging from problems with the assay design to inadvertently leaving out a necessary component from the reaction. Here are some recommendations:
-> Check the sequences of the probes and primers to be sure the bases and fluorophore/quencher modifications are all correct.
-> Run the failed qPCR on an agarose gel to check for the correct amplification product size.
- If you do not see an amplicon you can try adding more cDNA to the reaction mixture. If your amplicon is very GC rich, you can also try adding up to 5% formamide to the reaction mixture. You can also increase the Mg2+ concentration (in increments of 0.5 mM, up to 7 mM) but increased Mg2+ concentration can also cause nonspecific priming.
- If you do see an amplicon on the gel, check the real-time PCR instrument to make sure it is set at the correct wavelength to detect the right fluorophore and that it is set to monitor the fluorescence at the correct step of the PCR protocol which is after the elongation step.
-> Check the master mix components; it is possible that one of the components (such as the polymerase or probe) were inadvertently left out. We recommend that you repeat a failed experiment once to make sure it was not due to a simple mistake in the first round.
-> Check on the probe design. How long is the probe? What is the Tm of the probe? Probes must be around ~10°C higher than the primers so that the probe can hybridize before the primers. If the Tm of the probe is too low, it will not hybridize and no signal will be generated. In addition, try to avoid a probe with a G at the 5' end. The G can quench the fluorophore which may result in poor signal generation.
Would you like a copy of our qPCR Application Guide, that includes a troubleshooting chapter? You can follow the link to download or request a copy by mail: http://www.idtdna.com/pages/landing/qpcr/guide