Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens

PLoS One. 2012;7(8):e42341. doi: 10.1371/journal.pone.0042341. Epub 2012 Aug 1.


RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ∼10,000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Library*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction / methods*
  • RNA, Small Interfering / chemistry*
  • RNA, Small Interfering / genetics


  • RNA, Small Interfering

Grant support

Thermo Fisher Scientific employees' work was funded by Thermo Fisher Scientific. Some of the materials used in this study are products sold by Thermo Fisher Scientific. K. Jastrzebski and K.J. Simpson's contributions were funded by Australian Cancer Research Foundation, the Department of Industry Innovation and Regional Development, the Australian Phenomics Network, a Venture grant from the Cancer Council Victoria, the John T. Reid Charitable Trusts and Mrs Margaret Ross AM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.