Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector

Mellits, K. H., Pe'ery, T., Manche, L., Robertson, H. D., Mathews, M. B. (September 1990) Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector. Nucleic Acids Res, 18 (18). pp. 5401-6. ISSN 0305-1048 (Print)0305-1048 (Linking)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/2216712
DOI: 10.1093/nar/18.18.5401

Abstract

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

Item Type: Paper
Uncontrolled Keywords: Adenoviruses, Human/*genetics Cloning, Molecular Enzyme Activation Genes, Viral Genetic Vectors Promoter Regions, Genetic Protein Kinase Inhibitors Protein Kinases/metabolism RNA, Double-Stranded/*isolation & purification RNA, Viral/biosynthesis/genetics/*isolation & purification T-Phages/*genetics *Transcription, Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
organism description > virus > adenovirus
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: 25 September 1990
Date Deposited: 15 Mar 2016 19:33
Last Modified: 08 Nov 2017 16:49
PMCID: PMC332216
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32326

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