mirVana qRT-PCR with the Roche 1.5 LightCycler - (Jul/03/2006 )
I am trying to use Ambion's 'mirVana qRT-PCR miRNA detection kit' with the Roche 1.5 Lightcycler we have in the lab but keep getting inconsistent responses. I am only using it with the total RNA they supplied with the kit and the 5s and mir24 controls in duplicate at the moment to try and get it to work. However I keep finding that one or both of the 5s samples amplify, or one or both of the mir24 samples amplify. I cannot seem to get them all to amplify even though I'm fairly sure I'm following the protocol correctly. In fact using the protocol and kit for end-point pcr works fine. I was wondering if anyone else has experienced problems like this with this kit and lightcycler as Ambion are now suggesting that the two are not compatible but they couldn't give me a reason for why this would be? Any help is appreciated,
We've used that kit, mostly without any success. We also found inconsistant amplification across triplacates and the positive control (miR-24 and provided kidney RNA) only worked sometimes. Their tech support suggested running a dilution assay with total RNA from 1ng up to 250ng - again there was inconsistant amplification. We were using an ABI 7900HT realt time machine.