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RNAi: The Controls You Need

ost researchers agree that it is important to monitor RNAi induced knockdown at both the mRNA and protein levels. With an effective siRNA, you would expect to see reduction of both target mRNA and corresponding protein levels. mRNA reduction seen without a corresponding reduction in protein levels can indicate that protein turnover is slow--a different time point may be required for your biological assay. In contrast, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level as an miRNA. To save time and conserve reagents, you can analyze mRNA and protein levels from a single sample. The PARIS and mirVana PARIS Kits, which provide a simple protocol to isolate native protein and total RNA from the same sample, makes this particularly straightforward.


Use the Lowest Possible siRNA Concentration

Several reports indicate that siRNA concentrations of 100 nM or higher in mammalian cultured cells can lead to nonspecific changes in gene expression [2, 3]. Using 5-20 nM of a highly effective siRNA appears to minimize nonspecific effects. Well-designed siRNAs, such as Ambion's Silencer Validated and Pre-designed siRNAs, can often be used at even lower concentrations, further reducing the chance of eliciting off-target effects. Figure 1 shows a titration experiment in which various concentrations of Silencer

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Related biology technology :

1. RNAi: A How To for New Users
2. RNAi: Get the Whole Story
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5. Designing Controls for siRNA Experiments
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