I keep getting signals in the NTC.I am trying to detect HCV RNA in human serum samples using Taqman real time PCR method. In order to know the cause, I carried out an experiment with NTCs only (graph 2).
I don't know if it is a contamination or non specific binding ?
The curves do not look like amplification curves (except one curve in graph 1). The look like improperly background-corrected negative curves.
However, to make sure these are really representing negative curves, you should do two things:
1) amplify a standard with a (roughly) known number of about 10000 copies. This should give you a clear positive amplification curve with a Ct around 24. You can also amplify a dilution series. Also include several NTCs.
2) prepare a gel of the PCRs after amplification. I suggest doing a 6% PAGE. If you have the possibility, staining with a dye like SYBR Green or similar is prefferable over ethidium bromide (higher sensitivity).
Good luck.
PS: check the settings for background-correction of the amplification curves...
Hi Mohab, well if there is no template in your reactions it cannot be non specific binding. You probably contaminate your samples. qPCR is a very delicate and sensitive technique, you should use dedicated pipettes (and should wash them with soap once a week) dedicated tubes and plugged tips, a dedicated bench or hood where no DNA preps are done (and you never bring the amplified samples to).
The curves you show don't look very much like the classical exponentials, which are normally flat-like for at least 10-15 cycles and then start increasing. You cannot even set a baseline with such curves. Evidently there is some large non specific contamination that gives you a linear effect, the 5' primer only is working and the probe, so to produce a linear increase of fluorescence...
You probably need high sensitivity to detect the HCV in blood, so you should replace all your reagents, wash thoroughly the pipettes you are using (for the first time even with 1 M NaOH, to be sure to destroy all nucleic acid) and the bench/hood.
Good luck.
The curves do not look like amplification curves (except one curve in graph 1). The look like improperly background-corrected negative curves.
However, to make sure these are really representing negative curves, you should do two things:
1) amplify a standard with a (roughly) known number of about 10000 copies. This should give you a clear positive amplification curve with a Ct around 24. You can also amplify a dilution series. Also include several NTCs.
2) prepare a gel of the PCRs after amplification. I suggest doing a 6% PAGE. If you have the possibility, staining with a dye like SYBR Green or similar is prefferable over ethidium bromide (higher sensitivity).
Good luck.
PS: check the settings for background-correction of the amplification curves...
Thank you all for the answers.
I think contamination is highly unlikely as all the reagents are recently purchased and freshly prepared, even the water used from the kit but I will try to rule out that there is contamination as you suggested and see what will happen!
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