I am not getting any amplification for my target gene in real time PCR. The cDNA samples are fine since the amplification in positive control is fine. I have also obtained amplification for my target gene in semiquantitative PCR using annealing temperatures 60 and 62. Does any one has any idea where the problem is.
there may be many factors responsible including sub-optimal real-time PCR thermal profile, faulty reagents, low target copy number, etc. You may try doing a semiquantitative PCR using the real-time reagents and checking the amplicons on agarose gels. try to run a multi-copy target housekeeping gene transcript as your sample quality control for troubleshooting. optimize your reaction with a dilution series of target templates to check your assay sensitivity and so on.
Sibnarayan Datta the housekeeping genes amplifies fine with the same sample. the amount of template dna is same in real time pcr as used for semiquantitative.
In that case, you have to examine the sensitivity of your test gene primers.
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