qRT-PCR and DNA contamination

[Jeremy Coate]

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 <analia_alet from intech.gov.ar>
wrote:

> Hi Emad, for DNAse treatment I have employed TURBO DNA Free form Ambion
> (cat #
> 1907) and it worked well for me. Perhaps your problem is not the DNase.
> Perhaps you have two many DNA/ ug RNA. This kit, for example, removes up to
> 50
> ug DNA/ml RNA. If there is mor DNA in the sample, the manufactures advise
> to
> do a rigorous DNAse treatment, were you use more DNAse, adding it in to
> steps
> to the reaction mix and you duplicate the reaction time.
>
> Another thing to wonder: are you DNAse working well?
> Also you can optimize the digestion times with your owun sample and your
> own
> kit. Perhaps you need to treat the samples for more time. Remember that
> kits
> are standars, and sometimes you have to adjust them to your own conditions.
>
> Here it is the protocol we used:
>
> Reaction mix:
> 20ug RNA
> 10ul 10X Buffer
> 2ul  TURBO DNA-free
> up to 100 ul  water
>
> incubate 30 min 37ºC
> add 10 ul DNAse inactivation reagent
> incubate 2 min at room temperature (mix 2-3 times by inversion)
> ccentrifugate 15 min 10000g 4ºC
> SN to a new tube
> Store -80ºC
>
> Hope it works for you
> Good luck
> Best regards
> Analía
>
>
>
>
> Mensaje citado por methods-request from oat.bio.indiana.edu:
>  Hello,
>
> I am doing real time PCR for analysing my genes expression under different
> condition of my fungus. We use SYPR green. Actually I isolated RNA using
> both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on
> column following the kit's instruction. Unfortunately I could still detect
> a
> DNA contamination!! I treated the RNA again with DNase from NEB but still
> some traces of DNA exist. I tried to avoid the DNase treatment because I
> learned from some people who deal with RT-PCR to avoid DNase treatment and
> used Intron Span primers, and here is the big suffering, I used 18-20mere
> primers which locate just in the middle of exon borders and still these
> primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and
> still
> get some amplification, isn't that terrible? I thought to go to 3 bases at
> the 3' end of the primer but then you don't get any good prime which suit
> in
> this area!!?
>
> Can somebody please give me some hints or has any experience in such a good
> DNase which doesn't leave any DNA traces?! I am not optimistic in that.
>
> Emad
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>



-- 
Jeremy Coate
Dept. of Plant Biology
Cornell University
jec73 from cornell.edu
607-342-2679