qPCR using standard curve method - how to do correct setup? (Nov/13/2009 )
I'm quite new to qPCR and would like to get some help how to do a correct setup of the plate. I'm using SYBR green I togehter with the standard curve method.
My experiments are as follows, I have 7 different GOI and 1 HKG that I want to investigate for each of my 12 samples that are treated in different ways (+ diluted samples for standard curve, NTC etc). I want to have at least duplettes of each sample which makes it impossible to get all of the genes on one plate. So how should I do my plate setup?
Can I take one gene on each plate and be able to normalize them all against the HKG which has been run on a separate plate?
If this is ok, how about the threshold for the different runs, should they all be the same or should they be the optimal for each of the different genes?
I would be grateful for quick response since I don't really know what to do with my experiments.
I prepare all my samples in triplicates and at the same time so that my results are as reproducible as possible. My qPCR machine is a 48-welled one so if I have more than 2 samples I have to make multiple runs, which I do and it seems to work fine. So after I prepare my samples, I put them at 4oC until the time to run. People say that the polymerase in the SYBR green master mix is working at this temp, but I think that if you are able to run all your samples the same day, it should be fine. As to the threshold, it is better to adjust it so that it is the same for all your samples (for all the runs). Alternatively, if you do not trust this method, you can always run triplicates with control (reference) and the gene of interest each time: GAPDH and FOXO3; GAPDH and p53; GAPDH and cyclin D, for example (whatever genes you are testing for). This is much more expensive and time-consuming, but more reliable. Hope this helps
Admiral_Stuckov on Nov 14 2009, 07:55 AM said:
i second AS but i wud like to clarify a thing abt the polymerases in the master mix... in fact it is not at all active at 4 degrees cause they are blocked and need a temp of around 95 for 5 mins to be unblocked and activated!!!! so those people who say this are less informed about teh master mixes!!!
Admiral_Stuckov on Nov 14 2009, 03:25 AM said:
If I do the gene of interest and reference on each plate, like in your example: GAPDH and FOXO3; GAPDH and p53; GAPDH and cyclin D, do I then have to have the same threshold values for each of the runs to be able to compare FOXO3, p53 and cyclinD against eachother?