Effective expression of small interfering RNA in human cells

• MAY 2002 • VOLUME 19 • nature biotechnology 505 http://biotech.nature.com • MAY 2002 • VOLUME 20 • nature biotechnology 30 min at 37°C, either with or without preheating at 90°C for 5 min, followed by electrophoresis in a 15% denaturing gel. For detection of rev-EGFP mRNA, we used a 25-mer deoxyribonu-cleotide probe that was complementary to the EGFP mRNA of the rev-EGFP fusion protein. A 29-mer deoxyribooligonucleotide probe was used for detection of the GAPDH transcript. HIV-1 antiviral assay. For determination of anti-HIV-1 activity of the siRNAs, transient assays were done by cotransfection of siDNAs and infectious HIV-1 proviral DNA, pNL4-3 into 293 cells as described 15. Before transfection, the cells were grown for 24 h in six-well plates in 2 ml EMEM supplemented with 10% (vol/vol) FBS and 2 mM L-glutamine, and transfected using Lipofectamine Plus reagent (Life Technologies, GibcoBRL) as described by the manufacturer. The DNA mixtures consisting of 0.5 µg siDNAs or controls, and 0.5 µg pNL4-3 were formulated into cationic lipids and applied to the cells. After one, two, three, and four days, supernatants were collected and analyzed for HIV-1 p24 antigen (Beckman Coulter, Hialeah, FL). The p24 values were calculated with the aid of the Dynatech MR5000 ELISA plate reader (Dynatech Labs Inc., Chantilly, VA). Cell viability was also assessed using a Trypan Blue dye exclusion count at four days after transfection. Acknowledgments We thank Aaron Coleman for providing pIND and G. Pavlakis and A. Michienzi for the CMV-rev-EGFP vector. The authors also wish to thank Alessandra Poggi for help with some preliminary experiments.