Target Discovery and Validation Reviews and Protocols
Target Discovery and Validation Reviews and Protocols
Target Discovery and Validation Reviews and Protocols
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140 Matsumoto, Miyagishi, <strong>and</strong> Taira<br />
selection, <strong>and</strong> puromycin concentration. Initial cell number <strong>and</strong> puromycin concentration<br />
affect screening threshold because selection by antibiotics also affects<br />
knockdown efficiency.<br />
5. MasterMix for one 96-well plate should contain 2.5 mL of Opti-MEM I + 25 µL<br />
of LipofectAMINE 2000 reagent.<br />
6. Puromycin selection is critical because the transfection efficiency is not always 100%.<br />
7. MasterMix for one plate of 96-well plate: 2.5 mL of Opti-MEM I medium, 12.5 µg<br />
of poly(I:C) <strong>and</strong> 37.5 µL of FuGENE6.<br />
8. To select the positive clones correctly, appropriate negative controls should be<br />
included. As negative controls, we use a pcPUR hU6 vector for firefly luciferase<br />
gene <strong>and</strong> a pcPUR U6 vector for Renilla luciferase gene (30).<br />
9. Some proteins may have long half-lives <strong>and</strong> therefore phenotypic changes may<br />
require longer incubation. The cells could be analyzed approx 5 d after the transfection<br />
with siRNA expression library.<br />
References<br />
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(1998) Potent <strong>and</strong> specific genetic interference by double-str<strong>and</strong>ed RNA in<br />
Caenorhabditis elegans. Nature 391, 806–811.<br />
2. Zamore, P. D. (2001) RNA interference: listening to the sound of silence. Nat.<br />
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3. Bernstein, E., Caudy, A. A., Hammond, S. M., <strong>and</strong> Hannon, G. J. (2001) Role for<br />
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(2001) Argonaute2, a link between genetic <strong>and</strong> biochemical analyses of RNAi.<br />
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(2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured<br />
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8. Yang, S., Tutton, S., Pierce, E., <strong>and</strong> Yoon, K. (2001) Specific double-str<strong>and</strong>ed<br />
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