HELP. Multiplex RT-PCR with GAP as a housekeeping gene.

Dr. Duncan Clark Duncan at
Wed Feb 14 11:26:39 EST 2001

In article <3A8A850C.2D6818A3 at>, the eminent Thomas Mohr at
Thomas Mohr wrote
>Dear Colleagues,
>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....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382

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