Re: [DIYbio] Re: Gene silencing via miRNA

From what I have read, RNA interference is a much easier way to knock out the transcription of a gene without having to actually get in there and mess it up.

The protocols I have seen have you making dsRNA of a reasonable chunk of the gene and introducing it to the cell, where it gets cut up into miRNAs by dicer. There is a protocol for making dsRNA here.

This protocol highlights the difficulty for the hobbyist to deal with RNA: 

-RNA is very unstable and must be worked with quickly and stored at very low temperatures ( -70)

-to work with it requires a different type of sterility than normal (RNAase free, which you can read about here): baking your glassware, having access to either an ultrafiltration system and RNAase-free reagents or being able to use a carcinogen in your lab (DEPC)

-special kits for purification, although they are not particularly expensive ($5 / run for RNAeasy)

That said its also one of the most useful things in the toolbox, you can use it to examine expression or knock down genes with very high efficiency. I expect the first DIY project that actually changes the gene expression of a plant will use this. I will be quite impressed by whoever it is that does it.

On Sunday, March 17, 2013 8:17:16 AM UTC-5, Mega wrote:

Hi,

although I don't have any projects at the moment in this direction, I'm quite interested in miRNAs...


Can one design a mRNA like this:

If you have a promoter, there's the transcription initiation site where the promoter ends. Next to it you wouldn't attach any ribosome binding sites (because all you are interested in is the RNA, protein synthesis would be waste of energy). Then I think you would add the complementary sequence of the CDS (the first 20 nucleotides or so), excluding the ATG so it won't have a chance to silence other proteins.

Should work like this, right?

What makes it specifically interesting is that they are so short you basically could have them synthesized as primers, in this size without expensive purification...

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