I am attempting to optimize my RT-qPCR to run on Taqman Fast Virus Chemistry. My efficiency when running a standard curve is only about 80%. The amplicon produced is only approx 100bp and I get perfect efficiency using Qiagen One-Step under standard operating conditions. The standard assay runs uses 800nm primers/350nm probe. And is a 2+ hr long run. Rt 50C for 30 min, 95C 10min, then 40 cycles 95C for 15 sec and 60C for 1 min.
The fast assay run at 50C for 5 min, 95C for 20sec, then 40 cycles of 95C for 3 sec and 60C for 30 sec. The reason for the change is limited PCR machines in a diagnostic lab.
I have tried annealing temps of 56 to 62 (primer TMs 58C). This had no effect. I have also tried optimizing the primer concentrations: primer/probe 800nm/350nm, 500nm/250nm, 400nm/200nm, 300nm/150nm, and 200nm/100nm. All with little to no effect on efficiency. R2 values are consistently 0.998-9.
Please provide any thoughts or ideas because I feel like I'm spinning my wheels at this point.
Thanks in advance.
PS- I have done this with several other assays both RT and non-rt without needing to optimize using the Taqman Fast Virus and Fast Universal MMs.
Have you checked the Tm of the product? If it is a very high GC product, then 95C for 3 secs may be too low a temp for too short a time to get completely efficient melting. Do you have any way of checking whether the products of the fast and standard reactions are the same--just by running a gel for instance?
Thanks for the replies David and Laurence. The calculated Tm of the intended target is 74C with GC content of 58%. The target is only 76bp. I have not yet run a gel on the product or done a melting curve. We don't generally keep sybr green on hand but I am working on it. These were the next two steps on my troubleshooting list.
As for the cycle parameters, they were what was recommended by the product literature. The standard speed reaction used Qiagen One-Step kit, the fast protocol used the Taqman Fast Virus MM. I am all for adjusting denaturation time /temp for the initial step and/or cycle step so any suggestions are greatly welcomed.
Gel showed only one product exactly at the bp size it was supposed to be. The melt curve showed a single peak at ~72C.
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