Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA.
The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters. In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction. The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction. The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction. The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA. The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses. This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning. Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization.
Young
I D
ID
Department of Pathology, Queen's University, Kingston, Ontario, Canada.
Ailles
L
L
Deugau
K
K
Kisilevsky
R
R
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005260:Female; D005784:Gene Amplification / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000235:genetics*; D015347:RNA Probes; D016372:RNA, Antisense / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D009841:Oligonucleotides; D015347:RNA Probes; D016372:RNA, Antisense; D012313:RNA; D012321:DNA-Directed RNA Polymerases