RNA interference (RNAi) is a cellular biological process wherein small RNA molecules inhibit or silence gene expression. RNA is, of course, transcribed from genes and is the first step of gene expression. Transcription results in messenger RNA (mRNA) which is translated into protein. In the RNAi pathway, small RNA molecules interfere with gene expression by binding to mRNAs and inhibiting their expression. That is, the small or short RNAs function to prevent the translation of a specific mRNA into a protein and gene expression is down regulated.
From a mechanistic stand point, in the RNAi pathway double stranded RNA (dsRNA) corresponding to a specific gene sequence is broken down into small pieces of RNAs. These short pieces of RNA are referred to as small interfering RNAs (siRNAs). They are also known as short interfering RNAs or silencing RNAs.
Natural siRNAs are dsRNA duplexes of 21-23 nucleotides that trigger the degradation of their matching sequence mRNA, thereby preventing protein expression. In essence, degraded RNA is no longer able to translate into protein and in the RNAi pathway, gene expression is specifically silenced through the destruction of its cognate mRNA by specific siRNAs.
siRNA interference or silencing of gene expression through the RNAi pathway naturally occurs in many eukaryotes. However, today siRNA is most commonly recognized by scientists as a powerful research tool for reducing, knocking down, or silencing the expression of specific mammalian genes. In this technology, siRNAs are manufactured as synthetic oligonucleotide RNA duplexes designed to specifically target particular mRNAs for degradation. Robust design programs based on predictive algorithms are used to identify probable functional siRNA sequences. The synthesized siRNAs are then transfected into cells, for example by using transfection reagents or electroporation.
Knock down is assessed by PCR, western blot or by other readouts depending on the goals of the experiment. Of course, it is important to keep in mind that in order to assess knock down, researchers need a strategy for detecting gene expression. Although the most common methods are western blot and PCR, immunofluorescence microscopy, flow cytometry, functional activity, or other readouts may be used. The level of down regulation of a given siRNA experiment can be quite variable, ranging from complete silencing, partial inhibition, to total lack of down regulation.
Most researchers today prefer the convenience of ready to use siRNAs.
For this reason, MyBioSource offers a large portfolio of target-specific 19-23 nucleotide (nt) siRNA oligo duplexes optimally designed to knock down gene expression. The siRNA portfolio encompasses most of the common and even not so common targets researchers are seeking to knock down or silence.
From a technical standpoint, researchers often evaluate more than one target-specific siRNA oligo duplex to identify the optimal nucleotide (nt) sequence for knocking down expression of their gene of interest. This is because the gene silencing effects of a given nucleotide sequence may vary between cell types and model systems. The optimal sequence can often be determined empirically,by testing more than one predesigned and ready to use siRNA. Hence, many commercially available, ready to use siRNAs like those available at MyBioSource may be bundled together as a set.
In this regard, MyBioSource offers sets containing 3 different specific siRNA oligo duplexes to a given target.
Each siRNA in the set contain a different oligonucleotide sequence, designed to target and destroy different regions of the mRNA. These siRNAs are used for transient or short term knockdown of human, mouse or rat genes in vitro or in vivo. They can be transfected into cells individually to identify which one is most optimal for knockdown in their model system. Alternatively, they can be pooled together and transfected into the cells as a pool. Once knockdown effect is achieved with the pooled siRNA oligo duplexes, the siRNAs can be tested individually to determine the optimal siRNA for knock down.
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