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Quantification of siRNA Silencing Efficiency,,,Using the LightCycler System

Since the first description of RNA interference (RNAi) as a protection mechanism against invasion of foreign genes appeared a few years ago, RNAi has become a powerful tool for analyzing gene function in mammalian cells. RNAi is mediated by short double-stranded RNAs that are homologous to the suppressed gene. These small interfering RNAs (siRNAs) of two hybridized 21-mer RNA molecules (each containing 19 complementary nucleotides and 3′ terminal overhangs of two nucleotides) mediate sequence-specific mRNA degradation in mammalian cells [1].

To generate siRNA in vitro for transfection into cells, different methods such as chemical synthesis and in vitro transcription are used. Other methods apply vectors to generate siRNA in vivo within the cells. The method of generating siRNA and the siRNA sequence are both important factors for a successful knockdown experiment. Not all chosen siRNA sequences are able to repress gene expression with the same efficiency. A reduction of the mRNA level of > 90% is obtained with the most efficient siRNAs; others have little or no effect. Therefore, three or four siRNAs per target gene are usually tested to find the siRNAs that is most suitable, or in vitro transcribed RNA is digested using DICER enzyme to obtain a mixture of siRNAs. To make sure that an observed silencing effect on the protein level is specific to a used siRNA, the mRNA level of the target gene should also be measured. In addition, the mRNA can also be measured for genes where no specific antibody is available for the gene products. For proteins with a long half-life it is often easier to evaluate the efficiency of siRNAs on the mRNA level, because the knockdown effects are higher.

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