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

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, RNA...

Full description

Bibliographic Details
Main Authors: Brendon D Parsons, Anja Schindler, David H Evans, Edan Foley
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2009-12-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC2793519?pdf=render
id doaj-e5c1fe682ba842cf9a457e9e1b8a7e59
record_format Article
spelling doaj-e5c1fe682ba842cf9a457e9e1b8a7e592020-11-25T02:33:55ZengPublic Library of Science (PLoS)PLoS ONE1932-62032009-12-01412e847110.1371/journal.pone.0008471A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.Brendon D ParsonsAnja SchindlerDavid H EvansEdan FoleyRNAi 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.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.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.http://europepmc.org/articles/PMC2793519?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Brendon D Parsons
Anja Schindler
David H Evans
Edan Foley
spellingShingle Brendon D Parsons
Anja Schindler
David H Evans
Edan Foley
A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
PLoS ONE
author_facet Brendon D Parsons
Anja Schindler
David H Evans
Edan Foley
author_sort Brendon D Parsons
title A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
title_short A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
title_full A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
title_fullStr A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
title_full_unstemmed A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay.
title_sort direct phenotypic comparison of sirna pools and multiple individual duplexes in a functional assay.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2009-12-01
description 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.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.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.
url http://europepmc.org/articles/PMC2793519?pdf=render
work_keys_str_mv AT brendondparsons adirectphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT anjaschindler adirectphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT davidhevans adirectphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT edanfoley adirectphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT brendondparsons directphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT anjaschindler directphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT davidhevans directphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
AT edanfoley directphenotypiccomparisonofsirnapoolsandmultipleindividualduplexesinafunctionalassay
_version_ 1724811472442228736