loading control of RT-PCR - (Oct/18/2006 )
Hi all, when I do PCR to amplify the 1st cDNA of a targeted gene (say gene ABC), an internal control is needed, which is normally some house keeping genes like b-actin and GADPH. Now I have the primers for a gene ABC and b-actin, shall I put these 2 set of primers in a single PCR reaction or put them in separate PCR reaction?
The two different sets of primers should be put in different tubes and run at the same time...
Ideally, your control should be run at the same time as your gene ABC to serve as an internal control because each run is going to be slightly different each time. But if you can't do that because the optimal Tm of the primers are much too different, then I would either use a different housekeeping gene (cyclophilin, HPRT) or just make a note of it.
Does this help or did I give you an answer to a different question?
Both methods are OK. But, you need lots of optimization before you can get any good result from single tube reaction with two pairs of primers. You have to have primers with similar Tm, adjust amount of each primer pair in the reaction, make sure that there is no inter-primer hybridization, etc.
Thanks a lot. I will try both methods to see which one is better.