RT-PCR of an intron - possible plateau effect? (Jan/29/2006 )
I'm doing an RT PCR of an intron. within in the intron there is a pause site, and i have a set of primers before this pause site, and a set of primers after the pause site. The idea was, that before th pause site, i'd see a nice strong band, and after the pause site *hopefully* next to nothing. I've been getting really nice strong bands both pre-pause, and post-pause.
Now, if this is a real result, fine, great BUT, this is going against all my other results.
The protocol i'm using, has 40 cycles, which is a lot more than i would normally use (i got the protocol from another lab, which didn't have much success with it). Is it possible that what i'm seeing is the plateau effect? Perhaps there is less material post-pause, but because there are so many cycles, the difference between the two is diminished... or entirely wiped out?
I know, do another PCR with less cycles.... but i'd still like to have some other ideas on what could be happening here.
Just to note, it's all being normalised against a housekeeper.
I agree with your solution to do less cycles, the other thing I can think of may be that b/c you are looking at intron which is presumably only in pre-mRNA and this represents a smaller fraction of total mRNA and means you cannot use intron-spanning primers (of course! ) you may have more of a problem with small amounts of residual genomic DNA???
In other words you are amplifying gDNA before and after the pause site and there is enough of this template to mask the true result that would be seen if looking only at pre-mRNAs??
hope that helps ya... good luck...
What DNase are you using?
Might it be possible for you to use qPCR (I only like qPCR for this type of thing, especially with the possiblity of genomic contamination)?
I would also reduce the cycles down to 34 (at most).