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Question

Mention the complete steps involved in RNA interference (RNAi) in Biotechnology in an easy language.


Solution

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). 

To confirm that an observed phenotype is due to RNAi, it is also useful to perform time course and siRNA titration experiments.

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