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Recommendations for Successful siRNA Library Screens


By performing several RNAi screens with siRNA libraries, Ambion scientists identified five procedures that significantly improved siRNA library screen results.

Minimize cytotoxicity
Reduce siRNA concentration
Use individual, validated siRNAs, not siRNA pools
Correct for cell number
Note cell type differences

Here, we share our recommendations for achieving RNAi screening success. These guidelines will give your data more validity.


(1) Minimize Cytotoxicity

Because screening experiments involve monitoring a cellular phenotype, it is important to maintain cell health throughout the experiment. Long-term exposure to transfection reagents can reduce cell viability. Replacing the cell medium in wells approximately 24 hours post-transfection improves cell viability without reducing target gene expression. The optimal time for replacing medium varies by cell type and depends on cell sensitivity to transfection reagent and the rate at which siRNAs are taken up. Optimize these conditions by transfecting cells with Silencer GAPDH and Negative Control siRNAs; replacing medium at 4, 8, 24, and 32 hours; and measuring cell viability and GAPDH mRNA or protein for all samples at 48 to 72 hours post-transfection.


(2) R
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