Removal of double-stranded contaminants from RNA transcripts: Synthesis of adenovirus VA RNA₁ from a T7 vector

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 RNA₁ into a vector containing a 17 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 RNA₁ 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 RNA₁ and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.