An analysis and validation pipeline for large-scale RNAi-based screens

Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress r...

Full description

Saved in:
Bibliographic Details
Published in:Scientific reports 2013, Vol.3 (1), p.1076-1076, Article 1076
Main Authors: Plank, Michael, Hu, Guang, Silva, A Sofia, Wood, Shona H, Hesketh, Emily E, Janssens, Georges, Macedo, André, de Magalhães, João Pedro, Church, George M
Format: Article
Language:English
Subjects:
Animals
Bioinformatics
Candidates
Carbocyanines - chemistry
Embryo cells
Embryonic Stem Cells - metabolism
Gene expression
Mice
Oligonucleotide Array Sequence Analysis
Oxidative stress
Oxidative Stress - genetics
RNA Interference
RNA, Small Interfering - metabolism
RNA-mediated interference
Stem cell transplantation
Stem cells
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress resistance and found several candidates. We then developed a bioinformatics pipeline to prioritize these candidates incorporating effect sizes, functional enrichment analysis, interaction networks and gene expression information. To validate candidates, we mixed normal cells with cells expressing the shRNA coupled to a fluorescent protein, which allows control cells to be used as an internal standard, and thus we could detect shRNAs with subtle effects. Although we did not identify genes associated with oxidative stress resistance, as a proof-of-concept of our pipeline we demonstrate a detrimental role of Edd1 silencing in ESC growth. Our methods may be useful for candidate gene prioritization of large-scale RNAi-based screens.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep01076