3 replies to this topic

[joepog]

My lab have been having problems with QPCR. We perform knockdowns using Drosophila S2R+ Cells for 4 days using dsRNA (around 400-500bp) and should get an efficient knock down.

But when we come to analysing mRNA levels via QPCR we consitently get a fold increase in the range of 600-1000 times more in the cells treated with dsRNA.
Initially we thought this could be due to the QPCR primers amplifying our dsRNA this is not the case as they are both targeted to seperate regions of the gene. We know that the knockdown is having the correct effect via phenotypic analysis but this is a bit puzzling.

Has anyone seen this before or can anyone provide some insight as to what may be happening and what can be done to correct this?

Thanks

Joe

[WSN]

Try using oligo dT for 1st cDNA synthesis
A 1000 fold increase can only come from spiked external something.

[pcrman]

I would first want to rule out potential errors with qPCR. Have you treated RNA with DNase? Could be the amplification from DNA? Have you confirmed the change at protein level by Western? If errors can be ruled out and the induction of expression can be confirmed by Western, then this is something worthing further pursuit.

[mapplebe]

Hey, I am troubleshooting a very similar problem with RNAi in paramecium - I have no idea what is going on.  The phenotype has not been consistent in my case though so I am still working on my application method, but it is very frustrating!  Good luck to you!