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High-Content Imaging Analysis of the Knockdown Effects of Validated siRNAs and Antisense Oligonucleotides

Cancer Growth and Translational Genetics. Eli Lilly and Company, Indianapolis, IN

Shuguang Huang

Genomic and Molecular Informatics, Eli Lilly and Company, Indianapolis, Indiana

Michele Dowless

Cancer Growth and Translational Genetics. Eli Lilly and Company, Indianapolis, IN

Wayne Blosser

Cancer Growth and Translational Genetics. Eli Lilly and Company, Indianapolis, IN

Thomas Vincent

Oncology, Isis Pharmaceuticals, Carlsbad, California

Scott Davis

Oncology, Isis Pharmaceuticals, Carlsbad, California

Jeff Hodson

Oncology, Isis Pharmaceuticals, Carlsbad, California

Erich Koller

Oncology, Isis Pharmaceuticals, Carlsbad, California

Eric Marcusson

Oncology, Isis Pharmaceuticals, Carlsbad, California

Kerry Blanchard

Cancer Growth and Translational Genetics. Eli Lilly and Company, Indianapolis, IN

Louis Stancato

Cancer Growth and Translational Genetics. Eli Lilly and Company, Indianapolis, IN, l.stancato{at}lilly.com

High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments. (Journal of Biomolecular Screening 2007:775-788)

Key Words: mitosis • high-content screening • siRNA • antisense oligonucleotide • distribution analysis

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 775-788 (2007)
DOI: 10.1177/1087057107302675


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