I am doing my first qPCR experiments using TAqMan assays ( I know it is not the easiest start but we have the probes and the reagents already in my lab). Would you suggest a fool-proof protocol for someone who is setting this up for the very first time, it should be for evaluation the stability of my housekeeping genes (18S, GAPDH and ActinB). I would really appreciate any help.
Actually, Taqman assays are easier and shorter than SYBR green assays because you don't need to add a melting curve stage at the end of the protocol. The probe will bind your specific product if it's well designed. Besides, if the primers and probe are commercial, they will be very well optimized. I don't exactly know what you are asking, but I would suggest to do a optimization of the cDNA concentration to see in which range the PCR is working better. For this purpose, you can pool samples from your different groups and prepare different dilutions. If the taqman is not commercial you should also do a run with different concentrations of the primers and probe to test the efficacy. 18S will likely be quite far from the other genes.
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