HelpCorner - Gene Silencing

FAQ: When was gene silencing first described?

HelpCorner: In 2006, Andrew Z. Fire and Craig C. Mello received the Nobel Prize in Physiology and Medicine for their discovery of “RNA interference - gene silencing by double-stranded RNA”. “To their surprise, they found that double-stranded RNA was substantially more effective at producing interference than was either strand individually” [1]. This process, named RNA interference (RNAi), is strongly conserved in eukaryotes and presumably serves as a protection against viruses and genetic instability.


FAQ: What are the different RNAs used to silence genes?

HelpCorner: The technique of the long double-stranded RNA (dsRNA) used to silence genes in Caenorhabditis elegans [1] could not be demonstrated in mammals. These long dsRNAs trigger an unspecific interferon response in mammalian cells. To overcome this, shorter RNAs, 21-mer short interfering RNAs (siRNAs), have been chemically synthesized. The identification of more than 3,000 mature small RNA molecules, also called microRNAs (miRNAs), in species ranging from plants to humans, suggests that miRNAs are ancient players in gene regulation.


FAQ: What are the different classes of small RNAs?

HelpCorner: Many classes of small RNAs have emerged: siRNA, esiRNAs, miRNAs and piRNAs. They are defined in terms of their origin, structure, associated effector proteins, and biological roles. The three main categories are short interfering RNAs (siRNA), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs). These short interfering RNAs are present only in eukaryotes.


FAQ: Which classes of small  RNAs are the most broadly distributed?

HelpCorner: siRNAs and miRNAs are the most broadly distributed small RNAs, found in both plants and animals, where they are organized in phylogenetic and physiological terms; they are dsRNAs. In contrast, piRNAs are found primarily in animals, where they exert their functions most clearly in the germline; they are derived from precursors that are poorly understood, and appear to be single stranded.


FAQ: Have endogenous siRNAs been identified?

HelpCorner: More recently, endogenous siRNAs (­esiRNAs) have also been identified by sequencing short RNAs in mammalian cells (mouse oocytes).


FAQ: What are the differences between siRNAs and ­shRNAs?

HelpCorner: Since siRNAs are chemically synthesized, the design of efficient siRNAs is crucial for the success of the application. Both strands of an siRNA appear not to be equal during incorporation into its effector, the ribonucleoprotein complex (RISC). The thermodynamic characteristics of siRNAs and the accessibility of the binding region of the siRNA on the target RNA appear to be the two key points driving the efficient knockdown of the targeted gene. Even using strong design algorithms, siRNA can fail to down-regulate the gene of interest (probably due to accessibility issues). In addition, siRNAs are known to have a transient activity. They last for only several days, because of their degradation and dilution during cell division.

In 2002, an alternative to the chemically synthesized ­siRNAs, the DNA vector-based strategy, was presented to the scientific community. This new strategy permits a stable targeted gene inhibition. In this case, the altered gene function can be analyzed over an extended period of time. The new promoters used are optimized for the generation of large amounts of defined siRNAs. These short hairpin RNAs (shRNAs) are processed intracellularly by the endonuclease, called Dicer, into siRNAs, which go on to mediate gene silencing. In the most common system, the functional siRNAs are converted into a DNA sequence encoding the sense strand, with a loop, and the antisense strand. The catalytic component, cleaving the target RNA, has been identified as the protein Argonaut (Ago; Figure 1)

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FAQ: Is it possible to modify siRNAs?

HelpCorner: To increase the resistance of siRNAs to degradation, modified nucleotides can be used. These modifications can increase the half-life of the siRNAs, and also allow the introduction of markers such as fluorescent-labeled nucleotides. The hydroxy group, located at the 5´-end, must be phosphorylated to facilitate entry of the siRNAs into the RNAi pathway. Using an siRNA with a blocked 5´-end will lead to a loss of its inhibitory activity. The penetration of the siRNA into the cell can also be improved by employing a lipophilic component such as cholesterol.


FAQ: How can siRNAs be delivered?

HelpCorner: For cell-culture applications, transfection reagents are commonly used to deliver siRNAs into the cells. Some polymers, such as polyethyleneimine (PEI), are also used to deliver siRNAs. Electroporation and viral vectors are often used to integrate shRNA into embryonic mouse stem cells.


FAQ: Which products do you recommend to deliver ­siRNAs into the cells?

HelpCorner: For delivery of siRNAs, we recommend the use of the X-tremeGENE siRNA Transfection Reagent from Roche Applied Science. It is an optimized lipid-based transfection reagent that forms a complex with siRNAs, and with mixtures of siRNA and plasmid DNA. Another powerful transfection agent, the FuGENE® HD Transfection Reagent, has also been successfully used for siRNA knock-down assays [2].


FAQ: How should I proceed for the transfection?

HelpCorner: Reverse transfection can provide more definitive results than forward transfection. In reverse transfection, siRNAs are spotted directly into the cell-culture wells; this is followed by addition of the transfection reagent. After complex formation, cells are added, making reverse transfection ideal for high-throughput applications. This method is very effective when the E-Plates 16 and 96 (with microelectrodes at the bottom of each well) are used for continuous label-free cell monitoring with the xCELLigence System (Figure 2).

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FAQ: How can I analyze the phenotype induced by the introduction of the siRNAs?

HelpCorner: To determine the phenotype induced by the introduction of the siRNAs into the target cells, different techniques can be used. When cell proliferation is modified, end-point assays can be used to measure the changes, such as the Cell Proliferation ELISA, BrdU chemiluminescent or colorimetric kits. It is also possible to continuously monitor culture cells online, and to quantify the altered responses of cells transfected with siRNAs using the new xCELLigence System.


FAQ: How can I analyze the gene expression?

HelpCorner: The introduction of siRNAs targeting a given gene can also influence the expression of other genes. To monitor these changes, microarray and real-time PCR assays should also be conducted. Purify total RNA isolated at these time points using the High Pure RNA Isolation Kit. RealTime ready Focus Panels are ideal for gene expression profiling by real-time PCR using the state-of-the-art LightCycler® 480 System.


References

1. Fire A et al. (1998) Nature 391:806–811
2. Biochemica (2008) 1:21–24