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Is that normal the negative control showing signals?


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#1 xxcici

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Posted 15 April 2014 - 09:59 AM

For using 16s rRNA gene as a house-keeping gene for testing expression of other genes of a bacterium, is that normal the non-template-control giving a Ct value? Some people think it is normal.  



#2 bob1

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Posted 15 April 2014 - 12:47 PM

Depends on where the Ct value is and what it looks like - if it is over 35 then it may be some amplification that is non-specific and can be disregarded.  However, if the Ct is lower than that, then there is a good chance that one or more of your reagents or equipment is contaminated.



#3 ZebraEz

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Posted 15 April 2014 - 01:20 PM

I get that quite often, but it is just attributed to non-specific or background amplification. As long as the sample Ct is significantly lower that the NTC, then you can ignore this.



#4 Tabaluga

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Posted 15 April 2014 - 01:32 PM

Agree with the above posts; I was always told that it is safe if the Ct values of the negative control are very high and at least 5 cycles higher than the Ct of the sample with the lowest expression.


Edited by Tabaluga, 15 April 2014 - 01:38 PM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 xxcici

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Posted 16 April 2014 - 04:50 AM

Thank you so much for your reply. The Ct value for NTC is much higher than the sample. But I still do not understand what cause the amplification in NTC. Theoretically, there is no template, why there are amplification? 



#6 xxcici

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Posted 16 April 2014 - 04:53 AM

Depends on where the Ct value is and what it looks like - if it is over 35 then it may be some amplification that is non-specific and can be disregarded.  However, if the Ct is lower than that, then there is a good chance that one or more of your reagents or equipment is contaminated.

Thanks Bob. The melting curve of NTC is the same as sample, but Ct value of NTC is much higher than that of sample. 



#7 bob1

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Posted 16 April 2014 - 12:27 PM

I would say that you have a low level contaminant, which is probably PCR product... time to change your tips, tubes, and disposable reagents (water, primers)



#8 ZebraEz

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Posted 16 April 2014 - 01:20 PM

Have you had a look at the melt peak? It may be primer-dimer...

 

For the record, what are your Ct values for lowest concentration sample and NTC, respectively?

 

Note: If possible, could you post images of the amplification curves and melt peaks? It would make it a lot easier to understand the situation...



#9 xxcici

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Posted 17 April 2014 - 06:02 AM

I would say that you have a low level contaminant, which is probably PCR product... time to change your tips, tubes, and disposable reagents (water, primers)

Thanks Bob. 

the reagents used in the experiment has been changed with new sets. Tips are always changed and we used barrier tips. 

By searching from published papers, we see there are some mentioning the same problem for 16s rRNA amplification. So, I am wondering if there is something related to 16s rRNA itself, making NTC having amplification? 



#10 xxcici

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Posted 18 April 2014 - 09:55 AM

Here are the pictures of the melting curve and amplification plot. 

Attached Thumbnails

  • Melt Curve Plot_16S Problem NTC_1.jpg
  • Amplification Plot 16S Problem NTC_1.jpg


#11 Tabaluga

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Posted 18 April 2014 - 11:39 AM

You have just one melting curve peak, so looks like it is the same product that is amplified in your samples as in your negative controls, so I'd also go for the low level contaminant hypothesis. Presuming that the grey line at the amplification plot is your negative control, it has a Ct of 34, compared with a Ct of 8 for the samples I'd say it's OK. 


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#12 Poly44

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Posted 06 June 2014 - 03:42 PM

Hi xxcici,I was wondering if you managed to fix the problem. I am having the exact same problem, my amplification curves and corresponding melting curves look very much like the ones you posted. Were you able to determine if it a was a contamination issue at last?? 



#13 Mohamed 1984

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Posted 14 June 2014 - 12:22 PM

You can try to reduce the number of the cycles, the primer dimer always appear late in the run ( CT > 35 ). 

Another solution is to try to run it on Gel ( PCR) 



#14 dpo

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Posted 15 June 2014 - 10:23 PM

The polymerase used in your PCR can have a big influence on the level of positivity of your negative control. Remember that enzymes are produced recombinantly by bacteria and that, although companies try their best to remove all host DNA from the polymerase, low levels are almost always present. Since the 16S is strongly conserved, the primers used for the 16S of your bacteria of interest are also amplifying the DNA from the polymerase producing bacteria. There are several publications that try to tackle this problem (treat Taq with DNase, use specific low-DNA containing mixes, ...) but I don't have the reference at hand for the moment.

 

Two simple solutions to your problem: use a bacterium-specific control gene or disregard the problem (if the contamination is minor)

 

A more difficult solution: optimise your PCR by trying out different polymerases






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