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RNAi Tools for Epigenetics Research

By: Savita Bagga, PhD., BioFiles v7 n3, 2012, 17–18

BioFiles Volume 7, Number 3 — Epigenetics

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RNA interference (RNAi) is a relatively new technology that is revolutionizing the way researchers study gene expression. RNAi is a highly conserved gene silencing mechanism that occurs in response to the presence of double-stranded RNA. It's a pathway by which mRNAs can be down-regulated, aberrant RNAs degraded, and alien elements such as viral material, transgenes, and transposable elements can be suppressed.

RNAi works at the post-transcriptional, pre-translational level by targeting endogenous mRNA, with subsequent cleavage of targeted mRNA resulting in the effective knockdown of a given gene's activity. It is a powerful technique to study gene function in a rapid timeframe. Key to the RNAi processes are small interfering strands (siRNA), which have complementary nucleotide sequences to a targeted RNA strand. The siRNA “guides” proteins within the RNAi pathway to the targeted messenger RNA (mRNA) and “cleaves” them, breaking them down into smaller portions that can no longer be translated into protein and are eventually degraded.

Using this technology, researchers can specifically target/introduce double-stranded RNAs (dsRNAs) that are complimentary to known mRNAs into the cell to specifically destroy that particular mRNA allowing the study of molecular mechanisms.

Although all of the cellular functions and components of the RNAi pathway are not yet completely understood, the recent identification of a class of small non-coding RNAs called microRNAs in different organisms suggests the extent of RNAi-mediated gene regulation may be much greater than previously thought. microRNAs (miRNA) are single-stranded RNA molecules of 21–23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed but not translated into protein (non-coding RNA). Instead, they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. They typically differ from siRNA because they are processed from singlestranded RNA precursors. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. Animal miRNAs are usually complementary to a site in the 3'UTR; whereas, plant miRNAs are usually complementary to coding regions of mRNAs.

The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.

shRNA and siRNA Targets for Epigenetics

Sigma comprehensive shRNAi and siRNA product offerings consists of over 150,000 pre-cloned shRNA constructs targeting more than 15,000 human and 15,000 mouse genes. The library is available as gene family sets and gene collections related to epigenetics, stem cells, and cancer.

MISSION Human miRNA Mimics

MISSION miRNA mimics are small, doublestranded RNA molecules designed to mimic endogenous mature miRNA molecules when transfected into cells. miRNA are known to regulate gene expression in a variety of manners, including translational repression, mRNA cleavage, and deadenylation. MISSION miRNA mimics utilize a proprietary design to minimize potential sense strand off-target effects.

MISSION Target ID Library

The MISSION Target ID Library enables bench-top transcriptome-wide human miRNA and ncRNA gene target identification. It has an innovative dual selection system, which makes rapid whole transcriptome miRNA and ncRNA gene target screens accessible to any researcher with minimal reagent, time, or capital equipment expense.

Gene Silencing

Materials

     
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