Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens
نویسندگان
چکیده
منابع مشابه
Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens
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 trans...
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Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenoty...
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In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) de...
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BACKGROUND The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemicall...
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To successfully apply RNAi, the very first step is to design functional siRNA or shRNA sequences targeting specific genes. Many characteristics of functional synthetic siRNAs have been identified, but it remains questionable if these characteristics can be equally applied to the design of functional shRNAs. Our three-phase algorithm was developed based on the characteristics of functional siRNA...
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ژورنال
عنوان ژورنال: PLoS ONE
سال: 2012
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0042341