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10 fold dilutions show no RT-PCR product whatsoever - (Jul/25/2007 )

When I ran a RT-PCR rxn with pure DNA template (75ng) using SYBR Green I got a perfect amplification curve and a clear product band on a gel, but when I tried running a 10-fold dilution series (from 7.5ng to 0.75ng per reaction) I didn't get any product (besides some primer-dimer). I know RT-PCR sensitivity can go down to at least 1 ficogram of E.coli genomic DNA, which I'm working with, but I can't understand why I can't detect anything less than 75ng?? Has this happened to anyone else? Could it be the way I make up the dilution series- maybe I don't pipette the dilutions well enough to mix them uniformly? I dilute the DNA in RT-PCR grade water, the same tube I use in my Master Mix. Please help!


Careful just using the term "RT-PCR", different people will think you mean different things (Real-Time or Reverse Transcription) if they only see the title (it is clear from your text what you are doing). Yes, you should be able to amplify even with 0.75 ng dna in each well.

Here is what I would suggest. Perform an experiment with the DNA dilution at smaller fold decreases, such as 2x reduction each time, in new, small tubes (you said you used the same tube as the master mix, same type, or actually same tube?). Have you checked that your primers are good, as in performing a primer standard curve? You would do this by the same technique, consistent amounts of SYBR master mix in each reaction, consistent amounts of primers (I use 200 nM), and serially diluted source DNA. For example, if your primer annealing was 100% efficient, and you did 2 fold reductions, then each consecutive dilution would cross the cycle threshold one cycle later.

At what time did your 75 ng sample cross the cycle threshold? You are expecting the others about 3.5 and 7 cycles later, so if it was late and you did not run enough cycles you wouldn't have seen them. With 75 ng DNA, most genes should come up early though. Consistent pipetting is important too.


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