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Journal of Biomolecular Screening
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Article

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

Laurence H. Lamarcq1, Bradley J. Scherer1, Michael L. Phelan1, Nikolai N. Kalnine1, Yen H. Nguyen1, Tatyana Kabakova1, Xiaoyi Chen1, Marcia Tan1, Cynthia Chang1, Charina Berlon2, Roberto Campos-Gonzalez2, Guo-Jian Gao2, Stefan Golz3, Eugene S. Vysotski4, Andrew A. Farmer1*

1 Clontech Laboratories, Mountain View, CA.
2 BD Biosciences Pharmingen, La Jolla, CA.
3 Bayer Healthcare AG, Pharma Research--Molecular Screening Technology, Wuppertal, Germany.
4 Russian Academy of Science--Siberian Branch, Institute of Biophysics, Photobiology Lab, Krasnoyarsk, Russia.

* To whom correspondence should be addressed. E-mail: andrew_farmer{at}Clontech.com.


  Abstract

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 position-specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).

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

First published on February 20, 2006, doi:10.1177/1087057105284342

Journal of Biomolecular Screening 2006;11:236.

A more recent version of this article appeared on April 1, 2006

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