Signal In NTC - qPCR

[Masood]

Hi collages,

I would like to ask about the no template control that should always give no signal. In my case, I am trying to test the expression level of three genes and one housekeeping gene. Kindly note that all my reagents are new and are as follows :

• Applied Biosystems® Power SYBR® Green Master, also I have used Applied Biosystems® SYBR® Green PCR Master Mix, QuantiTect SYBR Green PCR Master Mix and KAPA SYBR® FAST qPCR Kit
• Ambion® Nuclease-free water
• Primers were reconstituted with Ambion® Nuclease-free water and also my primers have been used by many authors. ( Published primers sequences)


After using all the above mentioned reagents I have same signal in NTC. Could you please help?


Thanks all

Masood

[relaxin]

Do you mean you got signal from all primers of the four genes and all the master mixes?

Are you working on bacterial genes? If yes, the water may have bacterial DNA in it, even it is nuclease-free.

[Masood]

Hi again,

I am working with Human tissue, Kindly note that I have signal in all wells including RT- and NTC and the Ct values were between 26-30.

Please note that my primers are specific primers (expressed in a certain stage) for Humnan Protamin I and II.

best regards
masood

[relaxin]

I could not think of any other source of contamination. I hope other people can help you out.

[mchlbrmn]

Did you try an alternate water? If the water got contaminated, it would contaminate everything, including the primers. For PCR nuclease free isn't as important as clean.
Can the primers work on genomic DNA? That would make low level contamination more likely.
Do all the genes have the contamination?(!). That's strange, unless there's genomic contamination and all the primers work on genomic DNA.
This is SYBR: Does the contamination have the same melt peak as the targets?

[Masood]

Hi again.

Actually, I have used a new Ambion® Nuclease-free water bottle. The bottle has been opened under the PCR work station just to confirm and to make everything sterile.

Regarding the primers, I have used two sets of primers which have been used by my colleague and she has clean/ no signal at NTC.

In addition, all primers are intron spanning primer and DNAse was used to remove all gDNA.

The contamination or the signals were in all wells, RT-, NTC and in the GOI wells.

The confusing thing is that the melting curve is specific (one sharp peak).

I think I have to change the ABI 8-strip wells and covers; this is the only thing which I did not change!

Best regards
Masood

[mchlbrmn]

Wow, this is very tricky.
It's very odd if multiple targeted genes all give contamination, so even if PCR product got into the water the primers were resuspended in, it would be unlikely to contaminate with multiple genes, and genomic DNA contamination should not produce the size melt peak you see... actually it's possible that primers surrounding a small intron could coincidentally produce the same melt peak size. A gel might be needed to distinguish for sure, or a genomic DNA control could be run to see the product sizes there. I suppose it's also possible that a nonspecific band from genomic could produce the same size peak, but these are not very likely possibilities to happen for multiple bands from multiple primer sets.
I'm stumped. I'm tying to imagine you confusing the bottle of water with the tube of cDNA, or primer artifacts that happen to create products with the same melt point as the GOI -for multiple primer sets, but nothing really makes sense.
All I'm left with is to blame Ambion's water being spiked with cDNA.
Sorry I can't help.