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RNAi: A How To for New Users

rols are used for many different aspects of an RNAi experiment, often more than one control is required. For transfection optimization experiments, Silencer GAPDH siRNA is an ideal positive control. This siRNA efficiently silences GAPDH expression and its effects can be easily monitored by qRT-PCR or other methods at the mRNA level, or by Western blot or immunofluorescence at the protein level. In fact, Ambion provides a highly specific antibody for measuring these protein level effects.


Assay for RNAi Effect
There are several assays for measuring the RNAi effect. For understanding the biological effects of knocking down a target gene, cell based assays, enzymatic assays, array analysis, and countless other tools can be used. But before those assays can be performed, one needs to confirm that the siRNA is inducing knockdown of its intended target. siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation and for transfection optimization purposes is one that monitors target mRNA levels. The simplest, and arguably the best, assay for siRNA validation and transfection optimization relies on qRT-PCR to measure target transcript levels in gene specific siRNA treated cells versus negative control siRNA treated cells. Applied Biosystems TaqMan Gene Expression Assays, available for >41,000 human, mouse, and rat genes, are ideal for this
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