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J Biomol Screen. 2008 Feb;13(2):142-8. doi: 10.1177/1087057107312032. Epub 2008 Jan 23.

An efficient and fully automated high-throughput transfection method for genome-scale siRNA screens.

Author information

  • 1Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. namjin.chung@bms.com

Abstract

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.

PMID:
18216392
[PubMed - indexed for MEDLINE]
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