Frontiers in RNAi

Volume 1

by

Ralph A. Tripp, Jon M. Karpilow

DOI: 10.2174/97816080594091140101
eISBN: 978-1-60805-940-9, 2014
ISBN: 978-1-60805-941-6
ISSN: 2352-8400 (Print)



Indexed in: EBSCO.

RNA Interference (RNAi) arrived in the scientific community just over a decade ago, and has since blossomed with new connections being...[view complete introduction]
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Pooled shRNA Screening

- Pp. 58-78 (21)

Annaleen Vermeulen, Anja van Brabant Smith, Sarah B. Anderson, Roderick L. Beijersbergen and Kaylene J. Simpson

Abstract

Short hairpin RNA interference (shRNA) screens have earned their place in the technical repertoire of high throughput screening approaches by virtue of their broad applicability to targeting regular and primary cell types and the capacity to perform both positive and negative selection screens both in vitro and in vivo. This chapter focuses primarily on pooled shRNA screens, outlining the breadth of resources available, important library features and methods to establish effective transduction. We discuss assay development and optimization, followed by strategies for hit identification, principally using Next Generation Sequencing (NGS) approaches. Validation of any screen is essential and our collective experience guides the reader to consider a range of approaches towards confirming targets identified in the screen subsequently recapitulate the biological premise of the screen. We conclude with a thought provoking discussion on the future of shRNA screens, the challenges and the scope we can look forward to.

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