Gene silencing via miRNA

[Mega]

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...

[Josiah Zayner]

miRNAs are RNAs so primer synthesis is a no go because that is DNA. You can perhaps reverse transcribe a DNA element and then purify it and then inject/transfect it into a cell but this seems beyond DIYBio at the moment.

miRNAs go through extensive processing in the cell so I think most people tend to use shRNA and other things.

miRNA expression
http://www.origene.com/MicroRNA/

shRNA expression
http://www.genscript.com/siRNA_service.html?gclid=CLv6sZqdjrYCFYFDMgod-lMAEA

[Andreas Sturm]

I thougth of this: 

Promoter- UTR-miRNA- Terminator. 

RNA-Polymerase is "loading" at the promoter, and then it starts the translation. Some part of the MCS is also transcribed. Then there is the reverse complement of the DNA, so this gets transcribed into RNA. then there is still some UTR and then some polyA. 

So basically you get AGTGCTGCT(junk) - ATAGTCGCATCG (miRNA) - TCGTCGTC (junk)  The piece in the middle is complementary to the long protein coding RNA, and binds to it. 

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[Ben Hunt]

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:

[Andreas Sturm]

But why not introduce DNA into the organism which will then be translated into RAN anyway? So no need to directly work with RNA .

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[Emmette Hutchison]

shRNAs will also be more specific to whatever you want to silence, depending on how you design them.

Individual miRNAs can have hundreds of different targets that depend on the transcriptional context of a cell and miRNA targeting is still an area of active research. RNA editing can also change either the mature miRNA or the binding site in a target transcript as well.

Emmette

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