knockdown effect of RNAi by using qRT-PCR

Model Organism: Worms Methods & Reagents
1

Hello! I need your advice regarding RNAi.

I wanted to confirm the knockdown effect of RNAi by using qRT-PCR, but the expression level of my target RNA actually looked highly INCREASED. I got these kinds of results several times before when I used RNAi clones from Marc Vidal library. My guess is that double stranded RNA generated by RNAi bacteria was RTed and gave these opposite results as Vidal RNAi clones were generated from cDNAs. I think other worm scientists may have experienced similar problems, and I wonder whether there are any references that reported these problems. In addition, is there any experimental solution for this problem?

Thanks very much for your help!

2

This has come up before in the forum, and the consensus is as you suspect: your sample is full of dsRNA that you’re RT’ing and PCR’ing. You should make primers that bind in the 3’ UTR or use a smaller dsRNA construct that targets only part of the open reading frame and RT-PCR part of the message outside of the region covered by your dsRNA (preferably 3’ to your dsRNA, because of RNA-dependent RNA polymerase activity).