Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates.

RNA is playing an ever-growing role in molecular biology and biomedicine due to the many ways it influences gene expression and its increasing use in modern therapeutics. Hence, production of RNA molecules in large quantity and high purity has become essential for advancing basic scientific research and for developing next-generation therapeutics. T7 RNA polymerase (RNAP) is a DNA-dependent RNA polymerase of bacteriophage origin and it is the most widely-utilized tool enzyme for producing RNA. Here we describe a set of robust methods for in vitro transcribing RNA molecules from DNA templates using T7 RNAP, along with a set of subsequent RNA purification schemes. In the first part of this chapter, we provide the general method for T7 RNAP-based in vitro transcription and technical notes for troubleshooting failed or inefficient transcription. We also provide modified protocols for preparing specialized RNA transcripts. In the second part, we provide two purification methods using either gel-based denaturing purification or size exclusion column-based non-denaturing purification for isolating high-purity RNA products from transcription reaction mixtures and preparing them for downstream applications. This chapter is designed to provide researchers with versatile ways to efficiently generate RNA molecules of interest and a troubleshooting guide should they encounter problems while working with in vitro transcription using T7 RNAP.