RNAi technique

The chalcone synthase gene has also been silenced employing the RNAi technique. Silencing of this gene in the garden plant Torenia hybrida was successful resulting in flower colour alterations, results... 

The final proof was reached by the new and very powerful RNA inhibition (RNAi) technique (84). The technique is based on the fact that double-stranded mRNA is introduced into the cell by, e.g., electroporation. The cells recognize the injected RNA, as a viral invader, reduces it to shorter segments (20-23 base pairs) and disables protein biosynthesis from all similar mRNA templates. Introducing double-stranded mRNA which corresponds to either PAC a or PAC P inhibits the formation of the PAB as shown in interference contrast and fluorescence microscopy. At the same time, it blocked the step-up photophobic response (18). 

In a very recent study in potatoes, inhibition of LCYE accumulation was accomplished by an antisense LcyE driven by the patatin promoter and allowed rechanneling of lycopene toward the P-carotene branch of the pathway to produce up to 14-fold increased levels of P-carotene as well as up to 2.5-fold increased total carotenoids. RNAi and TILLING for manipulation of carotenogenesis have yet to be reported, but these new techniques for suppression of function and generation and selection of allelic diversity are likely to impact future research and production of varieties with enhanced pigment accumulation. 

After the selection of the specific techniques to be employed, one must determine how to quantify the genes associated with the phenotype of interest. The design of an assay that acts as a reporter or a molecular sensor can prove to be the most challenging step in designing a successful RNAi screen. The assay must be both highly reproducible and sensitive, without noise, and exhibiting minimal standard error. Traditional assay development techniques must be taken into account for all RNAi screens as they are for conventional compound-based screens. 

Techniques to overcome the lack of a negative (parental) control cell have become available through the use of RNAi, at least in cases where RNAi enters or can be expressed in the primary cells. High-throughput automated imaging systems have helped lessen the need to fully purify cells by allowing analysis at the single cell level. 

RNAi technology has a short yet very prolific history. In less than four years since the first demonstration that RNAi compounds can work in mammalian cells, the technique has found its way into virtually every relevant academic and industrial institution in the world. One of the reasons for such success most likely... 

Gene silencing techniques such as antisense, ribozyme, and RNA interference (RNAi) are also powerful tools to determine the transport activity of a specific protein. Flagenbuch et al. [162] have investigated the effect of coinjection of transporter (Ntcp or OatplalJ-specific antisense oligonucleotide on the uptake of BSP and taurocholate... 

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