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Mediating RNA interference in cultured mammalian cells. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RNA interference (RNAi) pathway.
RIPA buffer is a commonly used lysis buffer for immunoprecipitation and general protein extraction from cells and tissues. The buffer can be stored without vanadate at 4 °C for up to 1 year. [10] RIPA buffer releases proteins from cells as well as disrupts most weak interactions between proteins. [9] Recipe: [10] 1% (w/w) Nonidet P-40 (NP-40)
Small RNA (sRNA) are polymeric RNA molecules that are less than 200 nucleotides in length, and are usually non-coding. [1] RNA silencing is often a function of these molecules, with the most common and well-studied example being RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary ...
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic sulfonic acid buffering agent.It is one of the twenty Good's buffers.HEPES is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by aerobic respiration) when compared to bicarbonate buffers, which are also commonly used in ...
The siRNA guides the RISC complex to a specific sequence on the mRNA that is cleaved by RISC and, consequently, silences those genes. [2] However, without modifications to the RNA backbone or inclusion of inverted bases at either end, siRNA instability in the plasma makes it extremely difficult to apply this technique in vivo.
The RNase III Dicer is a critical member of RISC that initiates the RNA interference process by producing double-stranded siRNA or single-stranded miRNA. Enzymatic cleavage of dsRNA within the cell produces the short siRNA fragments of 21-23 nucleotides in length with a two-nucleotide 3' overhang.
An alternative concept to the usage of chemically synthesized siRNA for RNA Interference is the enzymatic digestion of long double stranded RNAs in vitro. In this case a cDNA template is amplified by PCR and tagged with two bacteriophage-promoter sequences.
The siRNA active component of Patisiran is formulated into lipid nanoparticles, which protect the RNA and facilitate its delivery to target tissues. The lipid nanoparticle formulation includes buffer components, as well as the lipid components DLin-MC3-DMA, Distearoylphosphatidylcholine, cholesterol, and the PEGylated lipid DMG-PEG 2000.