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RNAi: key technological aspects

Published on October 1, 2007 Reviewed on October 14, 2015   33 min

A selection of talks on Methods

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Welcome to today's discussion on RNAi key technological aspects.
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In brief, RNAi is a sequence-specific knockdown of genes at the messenger RNA level by double-stranded RNA. The ability of the double-stranded RNA to knock down genes of interest was first described by Craig Mello, Andrew Fire, and co-workers in 1998. This phenomenon was observed in the nematode C. elegans where the long double-stranded RNA could be administered by direct injection, soaking of worms in an RNA solution or by feeding them with bacteria expressing complimentary RNA strands.
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Early experiments that were aimed at defining the RNAi mechanisms revealed that in animal cells, long double-stranded RNA are not the RNAi triggering agents per say. Inside the cell, these molecules, the long double-stranded RNA, become a substrate for the enzyme called Dicer. Dicer cleaves the initial long double-stranded RNA into smaller RNA species, usually between 21 and 25 base pairs. And then, refer to a short interfering RNAs or siRNAs, composed of a sense and antisense strand. The antisense strand, often referred to as the guide strand of the siRNA, becomes incorporated into the RNA induced silencing complex or RISC which performs a cleavage of the desired target, the messenger RNA transcript of interest. Being fully cognizant of the biological role and their ability to avoid adverse cell death due to the short length, Thomas Tuschi and co-workers tested chemically synthesized siRNA molecules in mammalian cells and demonstrated for the first time their potent inhibition of the targeted gene in this model system. This discovery triggered the widespread use of RNAi indeed by specifically knocking down a single gene and monitoring the corresponding phenotype in the mammalian system. It has become possible to assign functions to those genes. When we consider the RNAi mechanism and its application to study in biological processes,