Web    easyRNAi.net   

Introduction

RNA interference (RNAi) is the process by which double stranded RNA (ds RNA) directs sequence -specific degradation of messenger RNA in animals and plant cells and some other organisms. In mammalian cells, RNAi can be acheived by 21-to 23- necleotide duplexes of small interfering RNA, called siRNA. Small interfering RNAs or siRNA, have gained much attention for their powerful ability to suppress gene expression without causing a non-specific cytotoxic response as longer dsRNA does. Small interfering RNAs siRNA or RNAi can be generated by chemical synthesis, in vitro transcription or intra-cellular transcription with a vector or PCR expression cassettes. The technique has been quickly used for functional gemomics in model oranisms, suppressing gene expression in mammalian cell lines and animals, prevention of human cells from virus infections.

History of RNAi

In 1990, Jorgensen and colleagues tried to enhance the colour of petunias by introduction of addtional pigment-producing genes into petunia. To their suprise, instead of the expected deep purple color, many of the flowers appeared patterned white or complete white.They named phenomenon observed as "cosuppression". At the same time, Alexander R. vean der Krol also observed similar phonotype when they introduced genes involved in flower pigmentation about quarter of plants demonstrated reduced floral pigmentatin, accompanies by dramatic reduction of expression of both the introduced gene and the homologous endogenous gene.

Guo and colleague produced the loss-of-function or gene-knockout phenotype in the offspring of the injected C. elegans by direct injection of antisense siRNA (par-1 gene) and a similar inhibitory effect on par-1 gene function was elicited by the injection sense siRNA. Cell 81:611, 1995

In 1998, Fire A and colleagues reported that double-strand RNA was at least tenfold potent as a silensing trigger than were antisense or sense RNA alone.

In 2000, 21 to 23 Nucleotide fragments hence siRNA from the dsRNA was demonstrated to guide mRNA cleavage Zamore P and colleagues.

Elbashir SM et al first reported that Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

Martinez J et al published the results demonstrating that Single-stranded antisense siRNAs are also effective to silence genes (5'-phosphorylated).

The RNAi Mechanism

dsRNA silences gene expression of the homologous gene. Upon cell entry, the dsRNA is cleaved by the nuclease Dicer into double-stranded small interfering RNAs (siRNAs) of 21 nt length with a two-base 3' overhang. These siRNAs are recognized by the RNA-induced silencing complex (RISC), and assembly of one siRNA strand into RISC is used to identify complementary mRNAs, thereby targeting them for destruction by a second nuclease in the pathway, Agounaute 2. In this manner, RNAi allows for the sequence-specific destruction of mRNAs expressed from both alleles in diploid organisms.

RNAi Applications

• RNAi as research tool
• Study physiology and diseases process in animal models
• RNAi as therapeutical strategy

siRNA made easy

General speaking, there are five methods for making siRNA:

1. Chemical synthesis of siRNA
2. In vitro transcription for siRNA
3. RNase III Digestion of dsRNA
4. siRNA expression plasmid or viral vector
   
   In most cases, the shRNA-encoding DNA fragment is made of two chemically synthesized 50- to 70-nucleotide-long oligonucleotides that are annealed and then ligated into a vector or assembled into a virus delivery system. This is a relatively straightforward method, although the synthesis of such long oligonucleotides is error prone.
5. PCR-derived siRNA expression cassette
   
   Alternatively, a Pol III promoter-shRNA template cassette can be assembled in a two-step PCR that uses shorter primers. The PCR product can be either cloned into a vector or directly transfected into cells.
   All of these methods, except creation of siRNA populations by digestion of long dsRNA, require careful design of the siRNA to maximize silencing of the target gene while minimizing the effects on off-target genes.
   Proper controls are needed for every RNAi experiment. A negative control that does not target any endogenous transcript is needed to control for nonspecific effects on gene expression caused by introducing any siRNA.

Delivery

In non-mammalian systems such as Caenorhabditis elegans and Drosophila, delivery RNAi into the cell is straightforward. For example, for delivery to Drosophila S2 cells, the dsRNA can be added directly to the media used to culture them. dsRNA can also be directly injected into worms or fly embryos. Most researchers use long dsRNAs previously proved to have an easily measured biological effect as a positive control, and dsRNA that does not target any transcript in the organism (e.g., dsRNA targeting luciferase) as a negative control.

In mammalian cultured cells, RNAi is typically induced by siRNA introduced directly or expressed as a hairpin structure from a DNA construct within the cells. Currently, the most widespread application of RNAi involves transient transfection of cultured mammalian cells followed by a downstream assay to monitor the RNAi effect. For this application, chemical synthesis is the preferred and most widely used method of siRNA preparation. siRNAs are easier to transfect than plasmids. More importantly, pre-designed, gene-specific siRNAs in a ready-to-use format are available, which makes this method the easiest and the most likely to succeed.

For many immortalized cell lines, transfection siRNA with a lipid- or amine-based reagent is the preferred option. Delivery into primary cells and suspension cells, however, can be problematic, if not impossible, using standard transfection methodologies. In these cases, electroporation using a specialized, gentle-on-cells buffer and optimized pulsing conditions generally results in very efficient siRNA delivery without compromising cell viability.

Virus infection has been widely used to deliver siRNA into culture cells and animals for both transient such as adenovirus and stable expression such as Retrovirus, Lentivirus and Adeno-associated virus. The expression has been regulated using Inducible Tetracycline, Ecdysone system and Cre system.

Measuring RNAi Effect

There are several assays for measuring the effect introduced by RNAi. For understanding the biological effects of knocking down a target gene, cell based assays, enzymatic assays, array analysis, and countless other tools can be used. But before those assays can be performed, one needs to confirm that the siRNA is inducing knockdown of its intended target. siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation and for transfection optimization purposes is one that monitors target mRNA levels. The simplest, and perhaps the best assay for siRNA validation and transfection optimization relies on qRT-PCR to measure target transcript levels in gene specific siRNA treated cells versus negative control siRNA treated cells. Applied Biosystems' TaqMan® Gene Expression Assays, available for >41,000 human, mouse, and rat genes, are ideal for this purpose.

Although it is necessary to monitor mRNA levels to validate siRNAs, most researchers also want to determine the extent of knockdown at the protein level. Western Blot, IF, ELISA and FACS analysis would be used once an suitable antibody is available. Some functional assays such as fluorescent or enzymatic colouring would also used. The best practice is to correlate siRNA, target mRNA, and target protein levels.

More RNAi Readings

Dicer -- Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18;409(6818):363-6.

shRNA -- Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 2002 Apr 15;16(8):948-58.

Silencing in murine cells -- Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1443-8.

In vivo p53 silencing -- Hemann MT, Fridman JS, Zilfou JT, Hernando E, Paddison PJ, Cordon-Cardo C, Hannon GJ, Lowe SW. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat Genet. 2003 Mar;33(3):396-400.

In vivo Neil1 silencing -- Carmell MA, Zhang L, Conklin DS, Hannon GJ, Rosenquist TA. Germline transmission of RNAi in mice. Nat Struct Biol. 2003 Feb;10(2):91-2. RNAi in heterchromatin silencing: Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science. 2002 Sep 13;297(5588):1833-7.

RNAi Glossary

Argonaute2 - A component of RISC (RNA-induced silencing complex), a 500 kD ribonucleoprotein complex with sequence-specific nuclease activity, that carries out gene-silencing during RNAi, purified in Drosophila cells and the sequence is homologous to rde-1, a member of the Argonaute gene family involved in RNAi in Caenorhabditis elegans. The gene was named as Argonaute2 (AGO2). AGO2-specific antibodies detected a 130 kD protein in the RISC complex. Disruption of the AGO2 gene by RNAi reduced the cells ability to perform gene silencing induced by double-stranded RNA.

Dicer - Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.

Interferon - A small and highly potent molecule that functions in an autocrine and paracrine manner, and that induces cells to resist viral replication. This term is related to RNAi because in mammals introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response.

Micro-RNA - Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt hairpin RNA precursors by Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals.

Off-target effects - Due to high concerntration as well as similarities between the off-target transcripts and the 5' ends siRNA may have suppression effects on genes that are not considered targets.

Post-Transcriptional Gene Silencing - Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation system designed to act as an anti-viral defense mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was initially described in plants and a related phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa

Ribozyme - Ribozymes are RNA molecules that act as enzymes in the absence of proteins.

RNA Interference - RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that small double-stranded RNA (referred as small interference RNA or siRNA) can induce efficient sequence-specific silence of gene expression.

RNA-Directed DNA Methylation - RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26-nt fragments. DNA sequences homologous to these short RNAs are then methylated and silenced.

RNA-Induced Silencing Complex - RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target.

RNAi Trigger - RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends.

Small Hairpin RNA - Small Hairpin RNA (shRNA) are produced as single-stranded molecules of 50-70 nucleotides in length and form a stem-loop structure in vivo. A 5- to 10-nucleotide loop connects the two complementary 19- to 29-nucleotide-long RNA fragments that create the double-stranded stem by base pairing

Small Interfering RNA - Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the cleavage and degradation of its cognate RNA.

Back to top