genes having different annealing temperatures in the same qPCR run - housekeeping gene and target gene have different annealing temperature (Jul/04/2011 )
In my experiments I have two treatment groups, one of which is the control. I want to see the expression levels for 3 genes between these treatment groups. However, my housekeeping genes and 3 target genes has different annealing temperatures. So in a single run, could you please guide me how to set up the temperature cycles with the genes having two different annealing temperatures. For example Tm of my housekeeping gene is 64C, whereas the Tm of my target gene is 73.5C. So, is it possible to run these two genes in a single run?
My second question is that what is the difference between using one housekeeping gene and two housekeeping gene in terms of experiment set up? I am trying to reproduce one of the paper's result and they use two housekeeping genes for normalization of target genes.
Thanks in advance
you can try to perform the PCR with the lowest temperature. Check that you don't get unspecific bands.
two housekeeping genes is better that one, (in case of your treatment would modify the expresion level of your housekeeping gene too, and this, you can not always know).