Efficientin vitrosynthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Abstract

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Development of a tRNA-dependent in vitro translation system

“…Plasmids were prepared for in vitro transcription as described previously (Brierley et al+, 1989)+ In vitro transcription reactions were carried out essentially as described by Melton et al+ (1984)+ For capped mRNAs, transcriptions included the synthetic cap structure m 7 GpppG (New England Biolabs)+ Product RNA was recovered by a single extraction with phenol:chloroform:isoamyl alcohol (49:49:2) followed by ethanol precipitation in the presence of 2 M ammonium acetate+ The RNA pellet was dissolved in water, and remaining unincorporated nucleoside triphosphates removed by Sephadex G-50 chromatography+ RNA was recovered by ethanol precipitation, dissolved in water and checked for integrity by electrophoresis on 1+5% agarose gels containing 0+1% sodium dodecyl sulphate (SDS)+…”

GOOSECOID inhibits erythrocyte differentiation by competing with Rb for PU.1 binding in murine cells

“…Proteins produced by in vitro transcription and translation RNA was synthesized as described (Melton et al, 1984). In vitro translation of RNA in rabbit reticulocyte lysates was conducted in the presence of [ 35 S]methionine as instructed by the manufacturer (Amersham LIFE SCIENCE, England).…”

Microsatellite deletions in the c-myb transcriptional attenuator region associated with over-expression in colon tumour cell lines

“…RNase protection assays were performed by modi®cations to the method of Melton et al (1984). Brie¯y, the a-[ 32 P]UTP labelled probe was synthesised, using a Message Maker Kit (Bresatec, Thebarton, SA, Australia), from a template that contained the human cmyb cDNA fragment (SacI 300 ± EcoRI 583 ) cloned into the pBluescript II KS + vector (Stratagene).…”