Quantitation of bovine TNF-alpha mRNA by reverse transcription and competitive polymerase chain reaction amplification.
A reverse transcription (RT) and competitive polymerase chain reaction (PCR) amplification technique was developed to quantify bovine tumor necrosis factor-alpha (TNF-alpha) mRNA. A bovine TNF-alpha cDNA insert containing plasmid was used to produce a 237-bp cDNA competitive template which, when PCR-amplified with primers designed to amplify bovine TNF-alpha cDNA, resulted in a 147-bp product distinguishable by agarose gel electrophoresis from the 378-bp TNF-alpha RT-PCR-amplified cDNA product. Dilutions of competitive template were co-amplified with constant amounts of total cellular RNA harvested from bovine alveolar macrophages (BAM). Products were separated in ethidium-bromide-stained agarose gels and relative amounts of TNF-alpha mRNA were determined by densitometric scanning of TNF-alpha cDNA and competitive template product bands on photographic negatives. Digestions with Pvu II and Sty I restriction endonucleases verified that RT-PCR-amplified TNF-alpha cDNA products were authentic. A time study indicated that TNF-alpha mRNA concentrations peaked 3 h after endotoxin and virus induction of BAMs.
Bienhoff
S E
SE
Department of Veterinary Microbiology, College of Veterinary Medicine, University of Missouri-Columbia 65211, USA.
Allen
G K
GK
D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000276:immunology*; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D016676:Macrophages, Alveolar / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000235:genetics
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha