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Rn ai and epigenetics sourcebook

Rn ai and epigenetics sourcebook



Make your RNA interference experiments simple, stress-free, and successful ...

Make your RNA interference experiments simple, stress-free, and successful
RNA interference (RNAi) is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects
of the loss of function of specific genes in mammalian systems. Once viewed as a technique used only by select laboratories, RNAi is now considered
essential for studying gene function. It has become a prominent tool for protein knockdown studies, phenotype analysis, function recovery,
pathway analysis, in vivo knockdown, and drug target discovery.
A brief history of RNAi
In the early 1990s, scientists first observed that RNA inhibited protein expression in plants and fungi. In 1998, Andrew Fire and Craig Mello, working
with Caenorhabditis elegans, discovered that double-stranded RNA (dsRNA) was the source of the inhibition, and they called this phenomenon
RNA interference (RNAi). While studies in C. elegans were encouraging, the use of RNAi was limited to lower organisms because delivering long
dsRNA for RNAi was nonspecifically inhibitory in mammalian cells. In 2001, shorter RNAs (siRNA) were shown to directly trigger RNAi in mammalian
cells, without evoking the nonspecific effects observed with longer dsRNAs. In 2006, just 8 years after the discovery of siRNA, the Nobel Prize in
Physiology or Medicine was awarded to Fire and Mello for their discovery, underscoring the importance of RNAi as an investigative tool.
How RNAi works
The molecules that mediate RNAi are short
dsRNA oligonucleotides, 21 nucleotides in
length, which are processed internally by
an enzyme called Dicer. The Dicer cleavage
products were first referred to as short
interfering RNA, now known as siRNA. RNAi
technology takes advantage of the cell’s
natural machinery to effectively knock
down expression of a gene with transfected
siRNA. There are several ways to induce RNAi:
synthetic molecules, RNAi vectors, and in
vitro dicing (Figure 1.1). In mammalian cells,
short pieces of dsRNA—short interfering
RNA— initiate the specific degradation of a
targeted cellular mRNA. In this process, the
antisense strand of siRNA becomes part of
a multiprotein complex, or RNA-induced
silencing complex (RISC), which then identifies
the corresponding mRNA and cleaves it
at a specific site. Next, this cleaved message
is targeted for degradation, which ultimately
results in the loss of protein expression



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