I’m having trouble to measure the levels of expression of a gene interfered by RNAi. I have repeated the RT-PCR three times and the levels of expression of
the interfered strain are higher than those of the wild strain. I have tried several primers of the gene and several housekeeping genes, but the
results are the same. (I am sure that the samples were not mixed at any time) Please, anyone know what may be happening?
I use 2000 worms in order to have enough RNA, could it be due to the efficiency of the interference? Thanks.
I guess the obvious questions are how you’re doing the RNAi, and what sequence you’re assessing by qPCR. If you’re using feeding, for example, you’re loading up the animal with a large dsRNA that may well cover the whole coding sequence of the gene - which you might then detect by RT-PCR.
I had similar issue in the past. I guess one of the reasons I could think of as HillelSchwartz suggested is that your real time primers might be binding to the dsRNA generated during RNAi or primers binding to sequence in the bacterial vector itself. and hence get more transcripts in your knockdown. one thing to make sure is that you do multiple washes before harvesting worms from the RNAi treated worms.I believe this way you might be able to get rid of bacterial dsRNA vector that is also a contaminating factor for real time PCR.
hi yamila,
just design a primer pair that bind in the 3’utr region, and everything is fine. the problem is that your rna is always contaminated with the rnai-plasmid of the bacteria.
cheers, oldcoffee