Using different primer and probe concentrations or different cDNA template dilutions, I always get pretty the same result. Once the signal is detected, the plateau appears fastly, so deltaRn is low. I use TaqMan polymerase, Cy3/Cy5/JOE probes, ABI 7500 amplificator. What should I check for?
Check the following RT-PCR Troubleshooting...
http://blog.biosearchtech.com/TheBiosearchTechBlog/bid/63622/qPCR-Troubleshooting
http://www.bio-rad.com/en-br/applications-technologies/real-time-pcr-doctor-gene-expression-quantification-experiments?ID=LUSOCICZF#gel1
Best luck.
The plot you are looking at has a log scale on the Y axis so you data are compressed and the "noise" at the start of the reactions is amplified. You need to look at the linear change in RFU vs cycle time to get a better idea of your amplification. What do your data look like overall? If you're primers have efficiency between 95-105%, good R^2 values for your technical replicates, and your controls all worked, then you are doing great.
Ayood Obaid Alfalahi has great links for you to read through for trouble-shooting. Good luck!
@Katie,
I respectfully diasagree. It is in fact the better idea to look at log(F) vs. cycle. Here you should be able to identify a clear (log-)linear phase in the curves. And it is exactly the problem here that these curves do not have such a linear part. This problem would be much harder to recognize in a plot of F vs. cycle. In this plot (log(F) vs. cycle) one can very clearly see that it is impossible to select a valid threshold value. One could thus not even determine the efficiency (at least not get a valid estimate)!
To my experience, simply using a different master mix it can completely change the game. This is the first I would try (try several! - ask companies for free test packs). Another easy thing to test is the PCR volume (if you run 5µl PCRs, it might help to try 20µl reactions). The next is to reconsider the probe system and to try to get rid of an internal calibrator dye (usually ROX).
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