通过逆转录和竞争性聚合酶链反应扩增对牛肿瘤坏死因子-α mRNA进行定量分析。
Quantitation of bovine TNF-alpha mRNA by reverse transcription and competitive polymerase chain reaction amplification.
作者信息
Bienhoff S E, Allen G K
机构信息
Department of Veterinary Microbiology, College of Veterinary Medicine, University of Missouri-Columbia 65211, USA.
出版信息
Vet Immunol Immunopathol. 1995 Jan;44(2):129-40. doi: 10.1016/0165-2427(94)05292-z.
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.
为了定量牛肿瘤坏死因子-α（TNF-α）信使核糖核酸（mRNA），开发了一种逆转录（RT）和竞争性聚合酶链反应（PCR）扩增技术。使用含牛TNF-α互补脱氧核糖核酸（cDNA）插入片段的质粒制备了一个237碱基对（bp）的cDNA竞争性模板，当用设计用于扩增牛TNF-α cDNA的引物进行PCR扩增时，产生一个147 bp的产物，通过琼脂糖凝胶电泳可与378 bp的TNF-α RT-PCR扩增的cDNA产物区分开来。将竞争性模板的稀释液与从牛肺泡巨噬细胞（BAM）收获的恒定总量的细胞总RNA共同扩增。产物在溴化乙锭染色的琼脂糖凝胶中分离，通过对照片底片上TNF-α cDNA和竞争性模板产物条带进行光密度扫描来测定TNF-α mRNA的相对量。用Pvu II和Sty I限制性内切酶进行的消化验证了RT-PCR扩增的TNF-α cDNA产物是真实的。一项时间研究表明，内毒素和病毒诱导BAM后3小时，TNF-α mRNA浓度达到峰值。
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