Tips on isolating low abundance genes using PCR - any advice? (Mar/05/2007 )
Hi all,
Based on what I've read about this gene, it is in low abundance even in the tissues where it is most highly expressed. Does anyone have any advice on how to isolate genes from cDNA when the target is in low abundance?
Cheers, Rob
My first guess would be to do lots of RT reactions, or use as much mRNA in the reaction as possible. I have been able to get single-copy virus target sequences out of 10^6 genomes, but only by doing large reactions.
It won't happen overnight, but it will happen.
It won't happen overnight, but it will happen.
Sorry Swanny, would you ming expanding on that? Not sure what you mean by 10^6 genomes and what sort of 'large reactions' are you talking about?
Sure thing, sorry I wasn't clear. I was looking for a single viral genome in 1 million human cells (10^6 genomes).
As for the large reactions, I was typically doing 200ul reactions. In order for the temperatures to equilibrate, I was using fairly long annealing and extension times. If your target sequence is large, you might want to try to make it smaller, or you might have to use a more conservative volume, like 100ul.