HELP. Multiplex RT-PCR with GAP as a housekeeping gene.
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Feb 14 11:26:39 EST 2001
In article <3A8A850C.2D6818A3 at utanet.at>, the eminent Thomas Mohr at
Thomas Mohr wrote
>currently we're trying to establish a multiplex RT-PCR with GAP as a
>houskeeping gene. Intended is the useage GAP primers plus a second
>primer pair (and maybe third). However, our GAP products require only
>22-25 cycles, compared to 30 to 35 cycles of the others. Coamplification
>with GAP primers added after 10-15 cycles lead so far to an almost
>complete quenching of the other product.
>Does anybody know of GAP primers fulfilling following condditions:
I don't see how changing primer pairs is going to help to much :(
The reason you need 22-25 cycles is purely because the copy no. of GAP
in your system is much higher than the copy number of whatever you are
trying to quantify.
So I reckon you need to find some other housekeeping gene that better
matches your other target copy no's, or add your GAP primers after 15
cycles or totally shift to real-time PCR with probes such that you can
then limit the concentration of GAP primers in your PCR, thereby
reducing the 'quenching effect'.
I don't know if Eric Leader from Ambion is still reading this newsgroup
but he has a lot more experience in this area and can maybe suggest
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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