Confirming SiRNA Knockdown - (Jun/13/2006 )
What are the methods that can be used to confirm knockdown other than western blot and RT PCR. I'm trying to do RT PCR but its not working very well and the protein levels are very low for detection by western blot.
thanks a bunch
Is the protien that u r trying to knockdown an enzyme? If so u could try some simple enzymatic assay.
Have a look at this paper, if it helps. Unfortunately its one of mine.
"Comparison of test systems for RNAinterference"
Biochem Biophys Res Commun. 2006 Mar 3;341(1):245-53.
RT-PCR is the simplest test to screen knockdown effect and you should get it work. For confirmation, something that doesn't involve amplification is better such as Western and Northern blotting. In my hand, RT-PCR is very consistent with Western so sometimes I just use RT-PCR to confirm. But sometimes RT-PCR is problematic such as DNA contamination and the target gene has pseudogenes careful DNase treatment of RNA samples and using proper controls in RT and PCR reaction can still yield reliable PCR results.
Thank you very much, this was helpful
A colleague of mine suggested bDNA to detect specific mRNAs within cell lysates. For more info check out the Panomics website. www.panomics.com
i heard that RACE is the thing to do. Anyone tried yet?
Have you tryed Western blot?