RNA Interference Complete Overview

This report on siRNA aims to summarize the current state of knowledge of the pioneering work of Fire, Mello and colleagues at the University of Massachusetts. The idea to control gene expression, perhaps go back to the early days of antisense approaches, whereby a single-stranded oligonucleotide could be designed, complimentary to the corresponding mRNA, in order to interfere with its translation. Likewise, in those days, stability and penetration were also issues to be contend with; both exo- and endonucleases diminished the effect of the antisense (AS) oligos; then various modifications were introduced to the diester backbone, including but not limited to sulfur, methyl, ethyl and other moieties, with the aim to extend the net half life of the AS oligos. Penetration wise, the AS oligos needed to be applied at high concentrations and the time of gene suppression was generally short lived; cationic based, transfection agents also were required. Amazingly, most short RNA’s that the majority of biological researchers dealt with, generally were indication of bad technique/degradation and were summarily discarded. Enter 1992, and it was found that indeed there was a natural gene inhibition mechanism, in which dsRNA was found to be used in Caenorhabditis elegans. Now it is also known that RNAi control of gene expression is a general mechanism found also in plants and mammalian organisms. This review will cover the known mechanism of Dicer splicing to produce 21-23 nts containing 2 nt base overhangs on both ends. This is followed by RISC interaction where the guide strand is prepared for final mRNA secission. The fine details occurring at RISC, where enzymes like Drosha, Pasha and others, interplay to finally produce the guide strand that then is ready to potently initiate mRNA cleavage, are still in the works. Chemical and in vitro approaches are also covered to give the reader the various alternatives to obtain inhibition of the gene of interest. Chemical modifications and delivery approaches are also discussed.  However, it is noteworthy to mention the phosphonoacetate approach, where the ODN’s themselves possess a unique penetration ability in a wide variety of human cell lines. The PDF document provides the following information:

1. INTRODUCTION
2. siRNA- A GENE SILENCER
3. COMPARSION OF GENE SILENCING APPROACHES
4. siRNA DESIGN
5. METHODS FOR SYNTHESIZING siRNA
   • CHEMICAL SYNTHESIS
   • IN VITRO siRNA SYNTHESIS
   • ENDOGENOUS EXPRESSION
6. CUSTOM siRNA SYNTHESIS
   • CUSTOM RNAi SERVICES AT BIO-SYNTHESIS
   • LOOKING FOR CONVENIENCE
   • PRODUCT PROFILE AND PRICING
7. siRNA TRANSFECTION AND DELIVERING OF siRNA IN TO CELLS
8. METHODS TO ACHIEVE HIGH TRANSFECTION EFFICIENCY
9. QUANTIFYING DOWN REGULATION
10. CHEMICAL MODIFICATION AVAILABLE FOR siRNA
    • TYPES OF MODIFICATIONS
11. CONTROLS USED FOR siRNA EXPERIMENT
    • POSITIVE CONTROL
    • NEGATIVE CONTROL
12. siRNA LIBRARIES AND siRNA SETS AVAILABLE IN THE MARKET
    • CUSTOM LIBRARIES OF siRNA AND siRNA SETS IN THE MARKET
13. APPLICATIONS OF RNAi TECHNOLOGY
    • RNAi in drug discovery and disease therapy
    • RNAi for genetic diseases
14. FUTURE PERSPECTIVES
15. REFERENCES