Is that normal the negative control showing signals? - (Apr/15/2014 )

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.  

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.

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.

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.

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? 

bob1 on Tue Apr 15 20:47:12 2014 said:

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. 

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)

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...

bob1 on Wed Apr 16 20:27:22 2014 said:

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? 

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