A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay

PLoS One. 2009 Dec 29;4(12):e8471. doi: 10.1371/journal.pone.0008471.

Abstract

Background: RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequence-specific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches.

Methodology/principal findings: To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes.

Conclusions/significance: Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypic penetrance for siRNA pools than for the parental individual duplexes. Thus, our data indicate that experiments with siRNA pools have a greater likelihood of generating loss-of-function phenotypes than individual siRNA duplexes.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Cell Death / drug effects
  • Gene Knockdown Techniques / methods*
  • HeLa Cells
  • Humans
  • Phenotype
  • RNA, Small Interfering / genetics*
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • RNA, Small Interfering
  • Tumor Necrosis Factor-alpha