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Design and Application of a shRNA 221transfected with pPUR V2 only and no DNA, respectively. Approx 20–30% of thecells on the plate should be transfected and the doubling time of U2OS cells isabout 24 h. Therefore, after 48 h of drug selection the plate transfected with pPURV2 alone should be about 60% confluent. There should be no live cells remainingon the plate transfected without DNA.2. A Bradford assay can be performed on samples in Laemmli sample buffer, providedthat not more than 1 µL of Laemmli buffer is added per 700 µL Bradfordreaction. Also, add 1 µL of Laemmli sample buffer to the reference Bradford reactions,containing 0, 5, and 10 µg of bovine serum albumin as standards.AcknowledgmentsThis work was supported by National Institute of Health R01 GM062281and the Leukemia and Lymphoma Society, Peter D. Adams (PDA) and theAmerican Federation of Aging Research, Rugang Zhang (RZ).References1. Hannon, G. J. and Rossi, J. J. (2004) Unlocking the potential of the humangenome with RNA interference. Nature 431, 371–378.2. Paddison, P. J., Silva, J. M., Conklin, D. S., et al. (2004) A resource for large-scaleRNA-interference-based screens in mammals. Nature 428, 427–431.3. Westbrook, T. F., Martin, E. S., Schlabach, M. R., et al. (2005) A genetic screenfor candidate tumor suppressors identifies REST. Cell 121, 837–848.4. Berns, K., Hijmans, E. M., Mullenders, J., et al. (2004) A large-scale RNAi screenin human cells identifies new components of the p53 pathway. Nature 428,431–437.5. Nicke, B., Bastien, J., Khanna, S. J., et al. (2005) Involvement of MINK, a Ste20family kinase, in Ras oncogene-induced growth arrest in human ovarian surfaceepithelial cells. Mol. Cell 20, 673–685.6. Kolfschoten, I. G., van Leeuwen, B., Berns, K., et al. (2005) A genetic screenidentifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121,849–858.7. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S.(2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing inmammalian cells. Genes Dev. 16, 948–958.8. Sambrook, J. and Russell, D. (Ed.) (2001) Molecular cloning: A laboratory manual.3rd ed., Cold Spring Harbor Laboratory Press, NY.9. Adams, P. D., Lopez, P., Sellers, W. R., and Kaelin, W. G., Jr. (1997)Fluorescence-activated cell sorting of transfected cells. Methods Enzymol. 283,59–72.10. Harlow, E. and Lane, D. (Ed.) (1988) Antibodies: A laboratory manual. ColdSpring Harbor Laboratory Press, NY.