Understanding RNAi

[swindoll]

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***Disclaimer, this is not for MCAT prep, but I figured this would be a good place to ask this question because you guys study a lot these days..
I need to learn more about RNAi for my next task in the research lab. I read the document that my professor recommended, but it's super dense with the technical vocab and is hard to understand. Do you guys have any pdfs or any good websites that would clarify RNAi for me? (The feeding technique in particular.) I watched Youtube videos, read some additional papers online, yet I still feel like I have some gaps in my knowledge.

Any recommendations will be appreciated!

Thanks!

 

[kraskadva]

If you're doing it in the lab, then you probably have a kit lying around for that. Go to the product page (on Sigma's, or whoever's) website and there's usually good rundown of the technique. Or just google Sigma's kits.

 

[blackmarble]

didn't study it for the mcat but from what i remember from my classes (i also got to attend a talk by andrew fire, one of the codiscoverers of RNAi once and it was pretty cool) is this:

RNAi is a method of preventing mRNA expression. The reason being is that cells have a defense mechanism in which it immediately recognizes and destroys double stranded mRNA in the cytoplasm (mRNA is naturally unstable due to the hydroxylated 2' carbon on the ribose sugar, so it degrades very rapidly via some intramolecular nucleophilic reaction with -OH as the nucleophile), probably due to some evolutionary adaptation which allowed it to recognize viral double stranded mRNA.

in any case, an enzyme/enzyme complex/multiple enzymes recognize double stranded DNA and degrades it in the cytoplasm, thereby preventing the mRNA from being translated.

So it's a good method in the lab to prevent gene expression and allows you to see what happens in the absence of a gene's protein product.

The mRNAi you expose to the cells is of course the antisense transcript. this allows it to base pair with the native sense strand in the cytoplasm. the antisense DNA strand is the coding strand of DNA in the nucleus that is transcribed by RNA pol and generates the sense mRNA strand, so that's why you inject the antisense mRNA-- so it'll base pair with the sense mRNA in the cytoplasm (I'm pretty sure this is right but you should double check).