title: Single-stranded RNA probes generated from PCR-derived DNA templates.
abstract: The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors.forename: K R
authors.initials: KR
authors.lastname: Bales
authors.forename: K
authors.initials: K
authors.lastname: Hannon
authors.forename: C K
authors.initials: CK
authors.lastname: Smith
authors.forename: R F
authors.initials: RF
authors.lastname: Santerre
mesh_terms: D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D051787:Myogenic Regulatory Factor 5; D016133:Polymerase Chain Reaction; D015347:RNA Probes / Q000096:biosynthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000096:biosynthesis / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015534:Trans-Activators; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D009124:Muscle Proteins; C497418:Myf5 protein, mouse; D051787:Myogenic Regulatory Factor 5; D015347:RNA Probes; D016372:RNA, Antisense; D015534:Trans-Activators; D014157:Transcription Factors