qRT-PCR and DNA contamination

I would reiterate a point that Brian made earlier in this string - negative
controls frequently exhibit some "amplification" in qPCR, but if the cycle
number is high it should not be a concern.

Even if you do have some residual genomic contamination that is amplifying
in the absence of cDNA (I'm guessing you are running controls in which you
do not reverse transcribe your RNA), it probably is not contributing to the
signal in your cDNA samples. My no-RT controls frequently show some
amplification (at high Ct's) that does indeed appear to be from genomic DNA,
but when I run the cDNA reactions on a gel there is no evidence of genomic
amplifcation. Presumably this is because the cDNA is in much greater
abundance than the residual genomic contamination. If the Ct of your
negative control is 10 cycles higher than your cDNA samples then the genomic
DNA is only about 0.1% of the cDNA abundance.

And, as Brian said, you frequently get a Ct value even if there is no DNA
template - its not unusual for me to see Ct's of 33 or so for my water
controls. But as long as your target templates are amplifying at much lower
cycles, whatever this artifactual amplifcation is should not be affecting
your results in any significant way. Of course, if you are getting
amplification in your controls at cycles similar to your targets, then you
do have a significant contamination problem, and the other suggestions
posted here apply.

Jeremy

On Thu, May 22, 2008 at 4:19 PM, Analia Alet <[Only registered users see links. ].ar>
wrote:




--
Jeremy Coate
Dept. of Plant Biology
Cornell University
[Only registered users see links. ]
607-342-2679