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Designing Controls for siRNA ,,, Experiments

es that up-regulation of the antiviral response may be a useful indicator of nonspecific siRNA effects. Although we do not yet routinely monitor the antiviral response in our own experiments, there are several published reports on how this response can be detected. One way is to assess levels of 2'5'-oligoadenylate synthetase mRNA (6). 2'5' oligoadenylate synthetase catalyzes the synthesis of 2'-5' polyadenylic acid, which activates the nonspecific ribonuclease RNase L. Another way is to monitor activation of the dsRNA-dependent protein kinase, PKR, which is involved in the type 1 interferon response. PKR phosphorylates the small subunit of the eukaryotic initiation factor 2-alpha (eIF2alpha), which results in nonspecific inhibition of translation. PKR activation can therefore be examined by monitoring the phosphorylation of eIF2alpha (7).

Monitor Both Target mRNA and Protein Levels
In siRNA experiments it may be beneficial to monitor both mRNA and protein levels for several reasons. For instance, mRNA reduction seen without a corresponding reduction in protein levels can indicate that protein turnover is slow. Furthermore, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level like a microRNA.

The use of RNAi has enormous potential for analyzing gene function, elucidating biological pathways, and identifying and validating potential drug targets. The RNAi field is
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