How to trace siRNA in cells - (Jul/03/2006 )
First question: I try to knock out my protein nm23 with siRNA. We want to see if this element could be involved in the signal transduction of my enzyme. What is the detection method of siRNA? Can you analyse the downregulation with semi quantitative PCR or must you necessarily do a real time PCR. Second question what is the best method (or kitt) to tranfect you’re siRNA when you using a rat celline. Where can you find a protocol for this experiment or depend this from celline?
1) people use labeled (Cy3, for example) siRNA to follow the distribution of siRNA in cells.
2) yes, you just have to do more PCR runs to get within the right ranges relative to your standards.
3) a lot. see general discussion on this topic above. You have to test which one is the best for your cell. I dont think any one can tell you with certainty: take this one, this will work for you.
I agree with genehunter.
One has to optimise transfections and choose suitable methods to analyse downregulation.
i used fitc labelled siRNA, works pretty well. i do not trust semi quant pcr and as for the rest: if there was a protocol for everything, would it still be research?...
Thats quite right !!!
Would take the fun out of research.