Workflow of qRT-PCR for siRNA - help please. (Mar/14/2010 )

I have another post on siRNA against Beta actin. and i am gonna look at in vitro cDNA synthesis after a trizol harvest. i've read about qRT-PCR on siRNA, but need help on the workflow. Do i need primers specific to my siRNA strand(S+AS) or how does this work?

Any suggestions or help is greatly appreciated.

why do you want to detect siRNA by qPCR or do you mean detecting beta-actin after RNAi?

dreww on Mar 14 2010, 10:26 PM said:

Assuming you wish to detect the level of knockdown for B-actin using qRT-PCR following siRNA transfection of an siB-actin oligo, you will most likely need to:
1) Seed your cells to be transfected
2) Transfect your siRNA of interest
3) Wait 24-72 hrs for knockdown of your target
4) Harvest your cells using trizol and isolate total RNA
5) Perform cDNA synthesis using random hexamers, etc.
6) Perform qRT-PCR using primers against your target (B-actin) that are specific for an RNA target (i.e. cannot amplify from a DNA template)
and also using primers specific for an unchanging house-keeping gene
7) Calculate the level of B-actin knockdown by normalizing against the expression of the house-keeping gene

Thank you Dr. teeth. that was my work flow. but instead of transfecting my siRNA, i got a vector to express my siRNA called pSilencer from ambion.
is there any way to detect the siRNA via qRT-PCR to make sure it's being expressed properly?

yes you can detect the expressed siRNA in a way similar to detecting mature miRNA. Usually it is not necessary to do that. Your target gene knockdown is a good indicator of siRNA expression

Thank you very much. it's good to hear that qRT-PCR is the best way to quantify knockdown. thanks everyone!