6 replies to this topic

[ooi]

Hi, I am using sybr green reagents to run my real time pcr. I used new filter tips and wipe all the pipettes with RNAsy water before use. The kit and primers are new, and I am using the supplied water.

I performed 40 cycles reaction. The house keeping gene; beta actin first show up after 15 cycle of the reactions and the gene of interest show up after 20 cycles. At about 30 cycles, the NTC of the beta actin gene seems to be expressing some specific genes. Some of the NTC of the gene of interest is showing up at about 35 cycles as well.

I did not add anything in the NTC. The NTC only contains the primers, water and sybr green reagent.

When checked under the melt curve, the peak of the beta actin NTC that I get is exactly the same peak as the template. It is not primer dimer peak. I find it so strange as I did not add in any templates in the control. And there is two peak in other primer's NTC. But the strange thing is the sample is only showing one peak when check under the melt curve.

I read from the forum that this might be because of the primer dimer formation and the primer dimer acts as the template for amplification. I did try to work out on the primer concentration. I tried a few range of the primer concentration. Apparently, it is still the same. The NTC is still showing up.

I do not have NTC contamination on the normal pcr under 30 cycles. However if I performed the pcr using the same genes for 40 cycles, the NTC will show up. The band of the NTC is exactly the expected band.

I am really exhausted with this NTC showing up after 30 cycles. Tried a few times, moving to another place to do the preparation... All did not work out. Please let me know if anyone can help me! Thank you very much!

[rajah]

Hi,

just curious: have you found any solution/ explanation for your problem? I sure do hope so since the post is old... I´m having the exact same problem at the moment. Any hints would be appreciated!

Thanks!!!

[dtae]

Unless you want to detect levels of gene expression 2^10x (or 1024x) less than when your target normally comes up, sounds like this is not really a problem.

The melting point of a product is determined by its base composition and size. It seems quite possible that primer-dimers could have the same melt point as your product. You could run it out on a gel to see if the size is also the same, or sequence it to see if it is a primer dimer of the target product.

D

[dtae]

also see http://www.protocol-...o-prepare-qpcr/
There someone suggests that it might be contamination of the primers.

[UBClabbie]

ooi, on 11 June 2010 - 08:32 AM, said:

Hi, I am using sybr green reagents to run my real time pcr. I used new filter tips and wipe all the pipettes with RNAsy water before use. The kit and primers are new, and I am using the supplied water.

I performed 40 cycles reaction. The house keeping gene; beta actin first show up after 15 cycle of the reactions and the gene of interest show up after 20 cycles. At about 30 cycles, the NTC of the beta actin gene seems to be expressing some specific genes. Some of the NTC of the gene of interest is showing up at about 35 cycles as well.

I did not add anything in the NTC. The NTC only contains the primers, water and sybr green reagent.

When checked under the melt curve, the peak of the beta actin NTC that I get is exactly the same peak as the template. It is not primer dimer peak. I find it so strange as I did not add in any templates in the control. And there is two peak in other primer's NTC. But the strange thing is the sample is only showing one peak when check under the melt curve.

I read from the forum that this might be because of the primer dimer formation and the primer dimer acts as the template for amplification. I did try to work out on the primer concentration. I tried a few range of the primer concentration. Apparently, it is still the same. The NTC is still showing up.

I do not have NTC contamination on the normal pcr under 30 cycles. However if I performed the pcr using the same genes for 40 cycles, the NTC will show up. The band of the NTC is exactly the expected band.

I am really exhausted with this NTC showing up after 30 cycles. Tried a few times, moving to another place to do the preparation... All did not work out. Please let me know if anyone can help me! Thank you very much!

likely caused by one of two things:
primer dimer formation - sol'n - redesign your primers or use hot start taq
contamination from the air - sol'n = reduce contamination by setting up your reaction away from other experiments (especially cloning), use a laminar flow hood to set up your reaction (use the UV light to break down contaminating DNA before set up, also expose your pipettes etc to UV light, just don't expose your template!)

to figure out which one, run a gel. if primer dimers are a problem you should easily see it on a gel. if its contamination, then you'll see a band at the expected size.

Edited by biotechgirl, 04 January 2011 - 04:35 PM.

[Prep!]

try the run preparing the samples in a LAF if not currently!!

[Trof]

I have often (in 30% of the cases of primers designed for/with UPL, but used without probe) seen (as I suppose) template-dependent primer-dimers (melting shows no dimers in the samples, but NTC goes up around 35 cycle with distinctly lower Tm than the sample) and I'm actually not doing anything with it. When there is not a dimer in my sample, and it's not a contamination, it doesn't affect the results. I know it's a bit different situation, than this one originaly described, but so you know.