Synthesis of Single-Stranded RNA Probes by In Vitro Transcription

Abstract In this protocol, we describe procedures for synthesizing RNA probes of high specific activity from DNA templates containing promoters for bacteriophage-encoded RNA polymerases. The DNA fragment to be transcribed should have previously been cloned into one of the commercially available plasmids containing bacteriophage RNA polymerase promoters. Alternatively, DNA templates can be synthesized in polymerase chain reactions (PCRs) using gene-specific primers whose 5′ ends encode synthetic promoters for bacteriophage-encoded RNA polymerases.