RNAi

RNAi :- 

is short for “RNA interference” and it refers to a phenomenon where small pieces of RNA can shut down protein translation by binding to the messenger RNAs that code for those proteins. 

Mechanism :- 

1) This process is related to normal defence against viruses and mobilisation of transposons. 

2) Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. 

3) RNAi has also been demonstrated in several vertebrate species but with lower efficiency.   4) Development of procedures for in vivo pr oduction of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.

5) RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers.

6) To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate.

Steps in RNAi :- 

Step 1 :- 

Obtain Effective siRNAs

To obtain gene silencing, potent and specific siRNAs are crucial. In addition, good experimental design dictates that at least two effective siRNAs be used in the experiment to confirm that the observed effects result from knocking down the gene of interest.

Step 2. :- 

Optimize siRNA Delivery to Maximize Gene Knockdown and Minimize Toxicity

Efficient, reproducible siRNA delivery is essential for successful RNAi experiments. The best siRNA delivery protocol provides good gene knockdown (i.e., effective delivery), while maintaining an acceptable level of cell viability (i.e., low cytotoxicity).

Negative control siRNAs are needed to identify potential non-specific effects on gene expression caused by introducing any siRNA. Easy-to-assay positive controls are also needed for optimization of transfection conditions, control of siRNA delivery, and as downstream assay controls.

Step 3 :- 

Test siRNA Silencing Efficiency

Because siRNAs exert their effects at the mRNA level, the simplest and most sensitive assay for siRNA validation relies on real-time RT-PCR to measure target transcript levels in cells transfected with gene-specific siRNAs versus negative control siRNAs.

Step 4 :- 

 Examine Biological Impact of Silencing Target Gene(s)

Assays that measure the effects of gene silencing include morphological, enzymatic, biochemical, and immunological assays. siRNAs affect target mRNA levels, but phenotypic changes are usually due to reduction of protein levels. siRNA-induced silencing at the protein level is typically measured by western blotting to correlate the observed phenotype with the level of knockdown induced (see sidebar, Western-SuperStar™ Immunodetection System).

Application :- 

Currently, most studies use RNAi as a tool for reverse genetics (identification of gene function), but the applications are numerous:

 i) disease control (viruses, bacterial diseases, parasites,genetic, tumors)

 ii) production of animals of commercial interest 

iii) control pests and diseases, introduce novel plant traits and increase crop yield.