Quantitation of bovine TNF-α 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-α) mRNA. A bovine TNF-α 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-α cDNA, resulted in a 147-bp product distinguishable by agarose gel electrophoresis from the 378-bp TNF-α 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 ethidiumbromide-stained agarose gels and relative amounts of TNF-α mRNA were determined by densitometric scanning of TNF-α cDNA and competitive template product bands on photographic negatives. Digestions with Pvu II and Sty I restriction endonucleases verified that RT-PCR-amplified TNF-α cDNA products were authentic. A time study indicated that TNF-α mRNA concentrations peaked 3 h after endotoxin and virus induction of BAMs.