QPCR primer Pairs - (Dec/04/2007 )
I am new to qPCR and I am having some problems understanding conditions required for my reactions. I am trying to analyse telomere length using QPCR. There are a couple of previous publications on this and I am using the same primers and conditions recommended by them.
They use telomere primers (tel) and single copy gene primers as a control (36B4). The reaction conditions are different for the 2 primer pairs and so they run them on different plates with a standard curve using control gDNA. I thought that it is always better to run both primer pairs on same plate if you can. The reaction conditions are as follows;
95oC 2 mins
for 25 cycles
95oC 2 mins
58oc 1 min
for 30 cycles
I don't understand why for the single copy gene there is no extention temperature, or why the annealing temp is for 1 min instead of 30"? And there is only a difference of 2oc for annealing temp so surely I can run them on the same plate? Does it even matter that they are on different plates?
many thanks, any help is much appreciated!
So you may want to e-mail the original authors, they would be best for answering this but one possibility is that these assays were designed independently, the 36B4 primers may have been optimized for those conditions and really in order to get data quickly the best bet is not to mess with something that is working, so they ran on two plates. You can definately try to accomodate both reactions, but if they work in your hands this way then I would leave it alone... There is not any issue with running the PCR for two different genes on two plates, what may be important for your qPCR system is that the standard curve for that gene is present on the same plate, but it doesn't matter if the two assays are run on separate plates.
Assuming the above, the absence of an extension temperature for the 36B4 gene may just be the way it was done initially, Taq polymerase can extend (of course somewhat depdt on the taq but in general) at temperatures less than 72C it is just a slower processivity and therefore a less efficient reaction, so it looks to me like they have simply combined the annealing and extension for this primer set for 1 minute rather than 30s each. However, if the efficiency calculated from your standard curve is acceptable or is canceled out by calculation (lightcycler) then I wouldn't mess with something that works, you could spend months optimizing the conditions to work on one plate or just run the experiment the way the other labs have... again, contacting the lab is the easiest way to find out the original reason for the difference...