Run-off RNA transcripts of in vitro transcription reactions have traditionally been analyzed using denaturing polyacrylamide gel electrophoresis (PAGE) and detection of labeled-UTP incorporated into the transcripts. Although multiple products from in vitro transcription reactions can be detected, quantitative analysis of RNA transcripts by denaturing PAGE is difficult. Ion-pair reverse-phase high performance liquid chromatography (IP RP HPLC) is a high-resolution technique that can separate nucleic acids based on size. Using denaturing conditions, RNA run-off transcript samples of mixed sizes can be separated and quantified. Several in vitro transcription systems using different RNA polymerases and various linear DNA templates with multiple promoters were assessed. IP RP HPLC improves the ability in detecting run-off transcription products, and establishes a better quantitative method for comparing transcripts of different length.

Ion-pair reverse-phase liquid chromatography analysis for separating and quantifying RNA generated via in vitro transcription reactions

249th National Meeting (2015)

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