No signal in Real-time but in normal PCR?! - (Sep/05/2007 )
I have a problem while trying to optimize my primers and probe for a gene in Real-time PCR (TaqMan).
I test my cDNA sample with my primers in normal PCR and I get nice bands on agarose gel. So I concluded that this cDNA batch does contain my gene-cDNA and that the primers are ok.
So I went on with real-time...
same cDNA, same primers, same concentration, addition of the probe (of course, it's TaqMan real time!), 40 cycles for normal PCR vs 45 for real-time...
and no results!!
No signal to be seen at all!
I loaded the real-time products on an agarose gel just to see, and it was very strange: the product stayed stuck into the pocket and didn't migrate at all. (1.5% agarose gel, theoretical product length approx 170bp)
I just mentioned that another gene was assayed at the same time. The real-time signal was low, and bands were visible.
Why do the products not migrate? What is that stuff sticking into the pocket??
It just can't be genomic DNA because I designed the primers so that the reverse primer overlays one exon-exon juntion, and the gDNA would be too long to be amplified in PCR (more than 3kbp).
Is it possible that there is no product at all and ethidium bromid alone does not migrate in electrophoresis?
I have no idea... if someone experienced this kind of thing or have an idea, please let me know ;-)
did you use a prefabricated real time pcr mastermix?
where is your probe from and how was it labeled?
what qPCR instrument did you use?
i think, if you have something in your reaction that inhibits gel migration of your product it will also inhibit the qPCR reaction. therefore it is not surprising that you can't detect any signal.
and since your reaction is working in the conventional pcr, there must be something wrong with your qPCR reagents.
I use a prefabricated master mix which works very well with the dozen of other genes I am investigating (Eurogentec MasterMix +, my primers and probes are also made by Eurogentec)
The probe is labeled with 3'FAM and 5' BHQ (Black Hole Quencher, from Eurogentec).
I thought as well about something wrong in the RT-PCR mastermix, but as it works very well with the other genes... ??? I am wondering why?? why now with this gene and not with the others??
Any other suggestion, or similar experience?
Thanks Ned Land