siRNA detection - Protocol for siRNA detection (May/25/2005 )

Hi all,

I am doing the research on applicationof RNA silencing in filamentous fungi. I used dual promoter vectors for suppressing eGFP gene and endogenous genes of filamentous fungi (Magnaporthe orzae). The results are very nice but I would like to confirm more that silencing phenomenon on the fungus is really caused by RNA silencing pathway. Therefore, I intend to do siRNA detection but I don't hav any experience about this. If you know, please give me an advice about this.

Thanks so much

Nguyen Bao Quoc

hi
best way is to resolve 15 to 50 µg of total RNA on a 15%polyacrylamide/7Murea gel transfer them on nylon membrane and probe them with a gamma32P-ATP T4polynucleotide kinase labelled probe.
Alternatively you can do dot blots.

Besides detecting the small sized siRNA by Northern as suggested by Fred, to prove that the effect is through the RNAi pathway, you can also knock down Ago protein using siRNA and see if the effect disappear after Ago is lost. I have done this in human cells. Although the window for detection is short (because Ago siRNA needs Ago itself) it is enough to prove something is dependent on RNAi pathway.

In my experiment, I used dual promoter system for functional genome-wide screening in filamentous fungi becase this system can overcome some limitations of single promoter system (haipin or intron hairpin dsRNA vector). I did transformation on wildtype strain and Dicer mutant strain. In Dicer mutant strain, there's no RNAi pathway so non-silencing phenomenon is very clear. That's enough to prove this system can induce RNA silencing pathway. I also did Northern blot to detect the concentration of mRNA and I got the degradation of mRNA on silenced samples as well as detected silencing construct in the genome of filamentous fungi.

Many thanks the suggestion of Fred and pcrman. Fred, could you please give me some instruction in detailed how to detect siRNA generally and dot blots as well

Thanks so much. I try to finish my experiement soon for my publication

Best regards,

Nguyen Bao Quoc