Mysterious -NRT control - (Aug/31/2011 )

It seems -NRT control problem will not leave me alone!

Currently my situation is....

1. Sample that is being used is total RNA and have been trizol extracted twice and turbo dnased twice (rigorous treatment)
As its bacterial RNA mixed in mammalian RNA, it has been microbenrinched and further amplified using Message amp kit, both from Ambion.

2. Previous SYBR green qPCR using same sample has shown no signal in its -NRT control for one set of genes that I am at looking at. However these few particular genes which I started looking into showed a singal in its -NRT control even though signals from first set of genes had way higher threshold cycle!
Eg. First set of genes had a signal at 17 cycle and no signal in -NRT control.
However! second set of genes which had a signal at 26 cycle had a singal in -NRT control at 32 cycle (why!!!).

3. Primers that are being used have already been screened using; dissociation curve analysis and dilution series for its efficiency and formation of primer dimers. Efficiency is within the acceptable range 90~110% and there is no formation of primer dimers. We use the same PCR program for all the genes.

4. Never had any signal in No template control.

I presumed that if the gene which had highest threshold cycle had no signal in -NRT control, all the genes which express at lower threshold cycle should have no signal in -NRT control... Guess this was a wrong presumption?

My samples are too precious for me to play around with (it's in vivo sample!)......

Please help me!!

A simple question -- Are you using aerosol resistant tips?

Just run your NRT and others as well on agarose and check whether you see any product in NRT, if you see over 100 may be there is some contamination, or if you see a detectable primer dimer only in the NRT and not in the experimental samples then you can leave of the NRT and analyse the rest. if primer dimer is seen in all including NRT then you got to redesign your primer....
This happens with SYBR, the same thing is also discussed elsewhere here...i couldnt find now, but the explanation was that it is a kind of template dependent primer dimers, you dont get such dimers if there a template in the reaction, which is mainly due to the Mgs in the mix which when left free forces the primers to form dimers...may be others here may give you better explanation..but just wanna let you know it is not something unusual esply with SYBR....

Yes, I do use aerosol resistant tips

I always try to carry out dissociation curve analysis. In this case, it has shown me a same single peak for both my sample and -NRT control. This should mean that primer dimer is not present in both my sample and -NRT control. Previously, I have checked my genes for primer dimers using agarose gel and it has shown me only one band (using different samples to this one though). Generally I would just presume that there is genomic DNA contamination in -NRT control. However.. it puzzles me that more highly expressed gene does not show any signal in -NRT control but a lower expressing gene will...

I'm going to try screening -NRT samples for all the genes again.. Hopefully it was just a one off thing but I dout that's the case...

jcho130 on Thu Sep 1 04:21:58 2011 said:

Do I understand it well, that you say you have some primers that amplify your sample at 17 and not the -RT, and some other primers that amplify the same sample with higher Ct but also the -RT at 32?
If It's correct and I'm not missing anything obvious then the problem is that the first primers don't amplify DNA but the second do. -RT amplification is dependent on the primers specifity for cDNA (intron spanning).