ArticleBMC biotechnology2009
Construction of siRNA/miRNA expression vectors based on a one-step PCR process.
Article in BMC biotechnology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
8 citing papers in PubMed.
- p53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression ofiScience · 2021Article
- Cloning short DNA into plasmids by one-step PCR.Thoracic cancer · 2020Article
- Design, simplified cloning, andMolecular biology research communications · 2016Article
- Size unbiased representative enzymatically generated RNAi (SURER) library and application for RNAi therapeutic screens.Nucleic acid therapeutics · 2015Article
- Construction and detection of expression vectors of microRNA-9a in BmN cells.Journal of Zhejiang University. Science. B · 2011Article
- A de novo mutation in NKX2.5 associated with atrial septal defects, ventricular noncompaction, syncope and sudden death.Clinica chimica acta; international journal of clinical chemistry · 2011Article
- MicroRNAs in adult and embryonic neurogenesis.Neuromolecular medicine · 2009Review
- Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA.Avicenna journal of medical biotechnologyArticle
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundRNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems.
resultsHere we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate.
conclusionThis novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries.
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