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Journal of Biomolecular Screening
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Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

Laurence H. Lamarcq

Bradley J. Scherer

Michael L. Phelan

Nikolai N. Kalnine

Yen H. Nguyen

Tatyana Kabakova

Xiaoyi Chen

Marcia Tan

Cynthia Chang

Clontech Laboratories, Mountain View, CA

Charina Berlon

Roberto Campos-Gonzalez

Guo-Jian Gao

BD Biosciences Pharmingen, La Jolla, CA

Stefan Golz

Bayer Healthcare AG, Pharma Research—Molecular Screening Technology, Wuppertal, Germany

Eugene S. Vysotski

Russian Academy of Science—Siberian Branch, Institute of Biophysics, Photobiology Lab, Krasnoyarsk, Russia

Andrew A. Farmer

Clontech Laboratories, 1290 Terra Bella Mountain View, CA 94043 andrew_farmer{at}Clontech.com

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

Key Words: RNAi • high-throughput screening • target validation • shRNA • reporter assay

This version was published on April 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 3, 236-246 (2006)
DOI: 10.1177/1087057105284342
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