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Journal of Biomolecular Screening
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An Efficient and Fully Automated High-Throughput Transfection Method for Genome-Scale siRNA Screens

Namjin Chung

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania, namjin.chung{at}bms.com

Louis Locco

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania

Kevin W. Huff

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania

Steven Bartz

Department of Biology, Rosetta Inpharmatics, a wholly owned subsidiary of Merck & Co., Inc., Seattle, Washington

Peter S. Linsley

Department of Biology, Rosetta Inpharmatics, a wholly owned subsidiary of Merck & Co., Inc., Seattle, Washington

Marc Ferrer

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania, marc_ferreralegre{at}merck.com

Berta Strulovici

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania

RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen. (Journal of Biomolecular Screening 2008:142-148)

Key Words: RNAi • RNA interference • siRNA • transfection • high-throughput screen • functional genomics • automation

This version was published on February 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 2, 142-148 (2008)
DOI: 10.1177/1087057107312032
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