Hello everyone,
as explained by the title of this subject, I would like to find a solution to a real time PCR issue. I saw many question about it, even on this site.
The thing is we already perform our experiments as described in the different answers that users provided here. So, let me give you some details:
- The subject is carried on DNA that we need to amplify for pyrosequencing.
We are working with Pyromark PCR Kit, revealing fluorescence thanks to SYBR Green. Our pre-PCR lab is separated from the PCR and post-PCR one. Primers are specific and sample are prepare either with a robot or manually.
So, when we saw this "ghost sequence" for the first time, we first thought about a contamination. In order to get rid of it we:
- Decontaminated pipettes with EtOH and then UV exposure.
- Changed our filtered tips.
- Changed Pipettes.
- Performed pre-PCR mix in different rooms and/or buildings
- Changed our PCR kit for the same model but with a different lot number.
- Changed for a PCR Kit provided with U N-Glycosylase
- Performed PCR with UNG incubation step
- Tried different waters (even with water for injection or UV irradiated water)
- Got new primers that we solubilized under white room conditions
- Tried a third PCR kit from a different supplier
Despite testing all these parameters, we still have and amplification at CT 30-32 in NTCs.
We performed melting curves that gave us "waves" from 50°C to 70°C and peaks at 78-81°C also corresponding to our sample fusion temperatures. Sample CT are around 12 to 17 (depending on the samples). Worse... the sequences on our figures are simply aberrant and do not correspond to anything (contamination between samples, contamination by natural products, by human...)
So my question is:
- Is it possible to get a very clean NTC, over 30-40 cycles?
- What could we try that we have not tried yet?
- If not possible... is there somewhere any publication, article, journal or whatever that could give us a proof that there is nothing significant in these NTC
Hi,
I used to have similar "positive NTC" problem in late qPCR cycles with one of my primers pair, but I chose to ignore those positive NTC's if the deltaCT between samples and NTC are bigger then 10 cycles (usually it is). I was using SYBR Green too. Here is an article discussing this kind of problem: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291693/
There is a suggestion in the article, that might be useful for you: "If all unknown samples record Ct values of around, say 18–25, and the NTC records a Ct of 39, then it is legitimate to ignore the very high Ct values recorded for the NTC and use the data"
One more option (that maybe you haven't tried yet) would be to optimize the concentration and ratios of primers. As explained in Thermo Scientific website, these positive NTC's are probably caused by primer dimers: https://www.thermofisher.com/cz/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/amplification-no-template-control.html
Another issue is possibly that your reactions are not in the linear range. Meaning if your sample CTs are 12-17, it may be too concentrated. We would have to dilute cDNA for 18S 1:10000 just to get the CTs in the 18-19 range, which is where they are linear. For all our other samples we routinely diluted them 1:50 or 1:100.
Kindly reduce your primer concentration. this will solve your problem. Probably the Ct values you are getting over 30 cycles are due to primer dimers. I believe your problem will get sorted out, without any negative effect over your reaction/results
Hi there! Thanks everyone for your quick response. I had a meeting with suppliers and other people concerned with my project and we are now planning to work over dimer concentration.
I feel really interested in the issue that Eugene mentioned. I firstly need more time to get that familiar with my equipment.
Anyway, thanks again for your time an help.
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