ArticlePloS one2012
Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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Who cites it
24 citing papers in PubMed, 40 citations in OpenAlex.
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- CRISPR: A Screener's Guide.SLAS discovery : advancing life sciences R & D · 2020Article
- Synthetic modeling reveals HOXB genes are critical for the initiation and maintenance of human leukemia.Nature communications · 2019Article
- Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.Genome biology · 2019Article
- Enhancement of the polymerase chain reaction by tungsten disulfide.RSC advances · 2019Article
- Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome.Journal of visualized experiments : JoVE · 2018Article
- Human immunodeficiency virus infection predictors and genetic diversity of hepatitis B virus and hepatitis C virus co-infections among drug users in three major Kenyan cities.Southern African journal of HIV medicine · 2018Article
- Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm.PloS one · 2018Article
- BRD4 inhibitors block telomere elongation.Nucleic acids research · 2017Article
- CRISPulator: a discrete simulation tool for pooled genetic screens.BMC bioinformatics · 2017Article
- Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens.Scientific reports · 2017Article
- Sources of Error in Mammalian Genetic Screens.G3 (Bethesda, Md.) · 2016Article
- Massively parallel high-order combinatorial genetics in human cells.Nature biotechnology · 2015Article
- The Power Decoder Simulator for the Evaluation of Pooled shRNA Screen Performance.Journal of biomolecular screening · 2015Article
- Malaria. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion.Science (New York, N.Y.) · 2015Article
- Article
- Size unbiased representative enzymatically generated RNAi (SURER) library and application for RNAi therapeutic screens.Nucleic acid therapeutics · 2015Article
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Authors and funding
11 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.