Problem with reference signal. - (Jan/04/2012 )
I have some problem with my reference (TBP). I ve made about ten reactions and in each of them I have signal in NTC for reference gene with Ct value about 28-33. There is no signal for target gene and no signal for target and reference gene in NO RT. Does anyone have any suggestions?
What type of assay, SYBR or Taqman? If SYBR, is the melting profile same as the samples or different?
I am working with Taqman.
It looks like a contamination. First try to change water, that's cheapest. Then new batch of primers. And clean your hood or bench and pipettes.
The reason why your RT- is negative may be, that the RT mix inhibits a bit and there are only a traces of contaminant (could be only TBP PCR product). Or you could crosss contaminate your NTCs but it's unlikely that this would be such reproducible.
On the other hand you probably can use your samples, if you have all RT- negative, because RT- is like a sort of negative control, that is even closer to your samples than NTC. But you should find the reson of Taqman assays NTCs giving a signal in any case.
Was your TBP NTC ever negative?
The Ct for reference gene is too high, most reference genes have lower ct values. when your reference gene gives ct of 33, it is possible that the gene of target, that most of the times has lower expression, gives ct values beyond measurement. Have you measured the amount of your cDNA? if you are diluting your cDNA, using higher amounts of cDNA may improve the results.
Hi, thanks a lot for advice. Some probes have negative NO RT but most of them are positive. Today I made reaction with different reference (GAPDH) and there is the same problem...
I my opinion there is no problem with water because in NTC I dont have signal from reference and target gene.
You don't have signal in NTC of what reference gene, other than TBP?
Anyway, try the water. It doesn't cost anything.
Taqman assay had to be so poorely designed to give false positive signal in NTC, that the contamination is still most likely. Question is where and how.
Did you ever get negative NTC in those genes you now see signals in? Or someone else. Just was the assay ever proven to have negative NTC or not?
No, I've never had negative NO RT and NTC for reference product. Diffrence between Ct for target gene and Ct for NTC and NO RT is about 5. I changed water and it doesn't work, there is still some product for reference. For some reaction I have reference signal only in No RT but for other one there is refence products in No RT and NTC...
I made reactions for the same gene with different refenece (GAPDH, TBP) and for GAPDH I have product in NO RT so I think there is some problem with GAPDH primers.... Reaction with TBP as a reference have negative No RT and NTC.
Didn't you write in first post you have positive TBP NTC? And now you write you don't. So it stopped being positive?
You can have signals in No RT, that's the traces of DNA, but you shouldn't have any in NTC.
Theoretically there may be dimer product, that can bind the probe as well, but this is really unlikely. I have never seen that. You probably can redesign the primers, while keeping the same probe, but these are usually designed together, that makes is complicated. And you can still have contamination in primers or probe. Probably a way to test this would be to put a primers (just them) or just a probe into NTC for other gene that went negative before and see if you will amplify anything.