Inconsistent signal in negative control- Taqman PCR

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Toasab
Toasab's picture
Inconsistent signal in negative control- Taqman PCR

I have a weird problem. I've got positive signals in my negative controls off and on. So, I did many tubes (4-9 tubes) of no-template-control from the same master mix running PCR at the same time and got positive signal in some tubes (2-4 tubes) and negative in the rest!!
The signal is good sigmoidal curve appeared at 35-up CT, and the negative is real nice and flat line. My internal control signal is normal and no indication of inhibition.
My PCR is real time PCR with Taqman probes (FAM for signal, and HEX for internal amplification control..so, it's kind of multiplex PCR).
Anybody can explain what happened?

Ivan Delgado
Ivan Delgado's picture
Hi Toasab,

Hi Toasab,

There are many reasons why you may be getting these results, including the possibility that it is simply the assay that is giving weird results that are being interpreted as being both positives and negatives. Of course one of the typical explanations would be to say there is contamination, which depending on how you setup your reactions it could explain these results.

Unfortauntely it is very hard to say what exactly is going on without knowing for sure what you are doing, the way you are setting up your reactions, the validity if your assay, and the exact nature of the amplification of your assay.

My main recommendation would be to obtain an assay you know works very well and run it side by side with this assay. If you still get both positives and negatives using this control assay in your negative control then there is a problem with your reagents and/or contamination. 

Good luck

Toasab
Toasab's picture
Deaar Ivan,

Deaar Ivan,
The assay was working fine for a while, no prob in the neg crl. And one day, the neg-crl started to give signal. First I thought it's contamination. So, I ordered new primers, probes, water, open new box of enzyme and buffer...but the problem still stay. I stopped working with this PCR for a while and now started again...still same prob.
But if it's contamination in any of my reagent...shouldn't all my neg-crl tubes turn out positive? What I think weird is that some are pos and some are neg...and they are just aliquots from the same mastermix oO!

Ivan Delgado
Ivan Delgado's picture
Hi Toasab,

Hi Toasab,

I hear what you are saying. Unfortunately there are many reasons why this could be happening. The new primers may have been synthesized with lower quality, leading to the assay not working as well as it used to. The fact that you are getting signals in some negative controls and not others is a strong indication of contamination, but then again that is very hard to prove. 

As I suggested in my previous post I recommend running another assay as a control side by side with this assay to see if the problem is assay specific. The type of problem you describe, in my experience, is due to the assay itself more likely than any other issue.

Toasab
Toasab's picture
Thanks Ivan, we'll keep

Thanks Ivan, we'll keep trying to get rid of this "contamination" problems...one of my suspect is maybe it came from the "autoclave" that we use to autoclave tips and tubes. It's shared with our target-organism (salmonella). Now the assay become more and more expensive, we have to buy pre-sterile tube, tips, and commercial water. Hope this works. Thanks for your help.

Toasab
Toasab's picture
Whowww...thought I found the

Whowww...thought I found the answer! I came across one paper with same phenomenon..they did hundreds of NTC and some came out positive ... same as mine. The possible cause is low level of contamination in PCR reagents, even commercial dNTPs and Taq polymerase has low amount of DNA contaminated out of the factory! And my PCR condition is happened to be very highly efficient and picked it up ^ ^ This is very good paper and will help people with contamination issue: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013042
I certainly will try some of the techniques.
Good luck to you all ^ ^

Maria das Graca...
Maria das Gracas Pereira's picture
NTC negative and positive amplification

Dear Toasab,I am facing issues with NTC the same way you described yours. NTC sometimes positive and sometimes negative. I use commercial RNase/DNase free water as a negative control and have also tried some alternatives (change gloves, different primers and probes), but the problem persists. Never faced this isssue before when targeting flippase and polymerase genes. Don't know if this has to do with primers and probes 16S rRNA as a target for bacteria identification. How did the assay turn out for you? Were you able to resolve it?Maria  

Ivan Delgado
Ivan Delgado's picture
Anytime. And contamination of

Anytime. And contamination of PCR reagents is indeed a possible cause of the problem. 

Good luck!

TAQMAIZE
TAQMAIZE's picture
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Hello Ivan,
I would like to ask you for suggestions in real time PCR experiments. 
I am getting some problems obtaining signal amplification in my negative controls in real time PCR.
I designed new primers and I got better results, but for some genes I am still getting signal in the NTC (No-template-control) as well as in NRT (No-reverse-transcriptase). The Ct values that I got are more than ten cycles from the Ct of my samples that contain cDNA. I do not think, it is genomic DNA contamination because the signal in both NTC and NRT is very similar, usually higher Ct values in NTC.
In the melting curve analyses (NTC), I got a peak higher than the threshold but lower than the sample that contains the cDNA. If I understood well, it means that the amplified product in the NTC is the same as in my sample containing cDNA. So even though the Ct value is more than ten cycles higher, can I keep on working with these primers or do I need to design new ones?
 
I also tried different primer concentration in order to see if the signal in the negative controls was reduced. However the best one is 10uM to get a good signal for my cDNA samples.
 
Did it happen to you before? I am trying to figure out where or when I get contamination in my samples. DO you have any suggestion?
 
Thank you very much.

Biju
Biju's picture
Hi Toasab

Hi Toasab
You should check the water used to reconstitute isolated RNA. It should be DNase RNase free and ideally purchased than being made in the lab. This could be one of the issue.
Biju Joseph