weird standard curve - (Jun/13/2006 )

Hi friends,
We are using qaigen sybr green kit for relative quantitation using GAPD as control gene. it was working fine in ABI 5700 however, we have now switched to ABI 7000. the main problem is that the standard curve for GAPDH has gone haywire. we use 400 ng of total RNA and use 6 dilutions each dilution 1/4th of the previous one. The Ct value for the first four dilution is perfect (a difference of two cycles for every dulution as expected) however, for the next dilution, the Ct value is equal to four cycles instead of two!! the subsequent dilutions have a correct Ct difference of two cycles. In addition, we are also observing contamination in our NT control and the melting curve shows same Tm as the specific product. We are also facing the same problem in some of the test genes we wish to quantify. Anybody else faced this problem with ABI 7000.

Hi,
we have the same standard curve problem, though we are using Roche Light Cycler. I mean, we sometimes get the same Ct values for the different concentrations of serially diluted standards. Since amplification itself seemed excellent, I was suspecting something wired had happened at the very beggining of amplification, or it could be because of a multigene family. It could be a common problem for real-time PCR users?

how long has it been since your machine was serviced?

the 7700 that I use gets crud in the wells that fluoresce...you can do a 'snapshot' of a blank block to see if that's the problem...can be cleaned with etoh, dH2O, and a Q-tip, plus lots of patience

also, how long has it been since the bulbs were checked?

I would try routine maintenance first

Thanks, we did complete routine maintanence during past few months, checked everything also got software upgrade to SDS 1.2.3. Actually yesterday, our reactions worked well both for GAPDH and control gene; perfect gradation for our dilutions!! But our problem of amplification in NTC well continues, with same Tm as in the other wells.

[quote name='Rat' date='Jun 14 2006, 06:21 AM' post='55372']
Thanks, we did complete routine maintanence during past few months, checked everything also got software upgrade to SDS 1.2.3. Actually yesterday, our reactions worked well both for GAPDH and control gene; perfect gradation for our dilutions!! But our problem of amplification in NTC well continues, with same Tm as in the other wells.

I am sorry to say this but its most lightly just a contamination of you Primer with template or pcr product. Just order new primer, use new water and new sybr mix and the problem should be solved. If not, run the NTC on a gel, it could also be that the primer form dimers with the same TM like your PCR product, but I realy think it's the first case.

As I wrote previously, we got occasionally weird standard curves with our real-time PCR system. We recently found out that we made two entry-level mistakes. Firstly, we used serially diluted standard DNA that was kept in polypropylene tubes for one or two weeks. I heard from a representative of Roche that a small amount of DNA adheres to polypropylene. This effect cannot be ignored when we deal with diluted samples. Actually, we tested this hypothesis by constructing two standard curves using a freshly diluted standard template and an old template that was diluted 10 days before. A standard curve made with fresh dilutions was indeed concentration-dependent and showed a good slope. In contrast, the other standard curve with a previously diluted template was odd. It was not correlated with dilution. Secondly, we overlooked primer-dimer formation with some specific primer pairs. We had tested the primer-dimer formation for these primers only by the melting curve analysis and by electrophoresis, but we did not run a negative control experiment without template DNA using those primer pairs.
I feel a bit ashamed to confess such beginners mistakes, but I hope our experiences are helpful to those who are relatively new to real-time PCR.