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Cited by 40RNA Interference and Gene DeliveryCRISPR and Genetic EngineeringMolecular Biology Techniques and Applications

Multiplexing siRNAs to compress RNAi-based screen size in human cells

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Martin, S. E., Jones, T. L., Thomas, C. L., Lorenzi, P. L., Nguyen, D. A., Runfola, T., Gunsior, M., Weinstein, J. N. +4 more·2007·Nucleic Acids Research
DOI 10.1093/nar/gkm141PMC PMC1885663
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Abstract

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study ( approximately 800 siRNAs, approximately 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

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  • The selected siRNA dose (20 nM) is used in routine in the authors' lab and is consistent with other works from the literature working with MCF10A or RWPE1.

    “This selected dose is used in routine in our lab (22) and is consistent with other works from the literature working with MCF10A (23) or RWPE1 (24).”

    From Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens (2018) · cites this paper as [23]

    The citation to this paper is attached to the MCF10A comparison, not the citing authors’ routine laboratory use or the RWPE1 comparison. The Methods (‘Transfection of synthetic siRNAs’; page unavailable) report 20 nM for paired siRNAs and controls, and report MCF10A transfections, but do not establish that the MCF10A experiments used a 20 nM total dose. The Results (‘Molecular validation of multiplexed siRNAs’; page unavailable) describe the illustrated MCF10A multiplex experiment at 60 nM total. (LLM, conf=0.84)

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Research works printed but not linked: 33

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Cited by

Sorted by citations

Authors
Martin, S. E., Jones, T. L., Thomas, C. L., Lorenzi, P. L., Nguyen, D. A., Runfola, T., Gunsior, M., Weinstein, J. N., Goldsmith, P. K., Lader, E., Huppi, K., & Caplen, N. J.
Affiliations
  • National Institutes of Health (US)
  • Qiagen (Germany) (DE)
Corresponding author
Caplen, N. J.
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2007-03-29
Volume
35
Issue
8
Pages
e57-e57
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkm141
PMC
PMC1885663
OpenAlex
W1974512564
Cited by
40
References
1
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2022: 2 · 2020: 10 · 2018: 2 · 2017: 1 · 2015: 2 · 2013: 3 · 2012: 1
FWCI
1.33
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Cancer Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Keywords
RNAi screen, gene silencing, cell viability, breast cancer cell lines, positive control, functional genomics
MeSH terms
Cell Survival, Humans, Gene Library, Oligonucleotide Array Sequence Analysis, RNA Interference, RNA, Small Interfering, Cell Line, Tumor

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