ArticleBMC biotechnology2006
PCR-based generation of shRNA libraries from cDNAs.
Article in BMC biotechnology, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 28 citations in OpenAlex.
- A multi-shRNA vector enhances the silencing efficiency of exogenous and endogenous genes in human cells.Oncology letters · 2017Article
- Size unbiased representative enzymatically generated RNAi (SURER) library and application for RNAi therapeutic screens.Nucleic acid therapeutics · 2015Article
- Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing.Proceedings of the National Academy of Sciences of the United States of America · 2010Article
- Heat shock protein 27 mediates repression of androgen receptor function by protein kinase D1 in prostate cancer cells.Oncogene · 2009Article
- Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.PloS one · 2009Article
- Construction of siRNA/miRNA expression vectors based on a one-step PCR process.BMC biotechnology · 2009Article
- Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells.Biochemical and biophysical research communications · 2008Article
- Beta-catenin mediates alteration in cell proliferation, motility and invasion of prostate cancer cells by differential expression of E-cadherin and protein kinase D1.Journal of cellular biochemistry · 2008Article
- Angiopoietin-1 targeted RNA interference suppresses angiogenesis and tumor growth of esophageal cancer.World journal of gastroenterology · 2008Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
Abstract
backgroundThe 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 chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.
resultsWe report here an improved method for constructing genome-wide shRNA libraries enzymatically. The method includes steps of cDNA fragmentation and endonuclease MmeI digestion to generate 19-bp fragments, capping the 19-bp cDNA fragments with a hairpin oligonucleotide, and amplification of the hairpin structures by PCR. The PCR step converts hairpins into double-stranded DNAs that contain head-to-head cDNA fragments that can be cloned into a vector downstream of a Pol III promoter.
conclusionThis method can readily be used to generate shRNA libraries from a small amount of mRNA and thus can be used to create cell- or tissue-specific libraries.
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Registered trials
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.