Development of methods for quantitative comparison of pooled shRNAs by mass sequencing

Hirotoshi Hoshiyama, Jun Tang, Kimberly Batten, Guanghua Xiao, Jean Marie Rouillard, Jerry W. Shay, Yang Xie, Woodring E. Wright

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.

Original languageEnglish (US)
Pages (from-to)258-265
Number of pages8
JournalJournal of Biomolecular Screening
Volume17
Issue number2
DOIs
StatePublished - Feb 1 2012

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Keywords

  • RNA interference
  • emulsion PCR
  • half-hairpins
  • in vivo screening
  • multiplex assays
  • parallel sequencing
  • shRNA libraries

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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