摘要
本文尝试了以 cRNA 为内在参照物,以逆转录—DNA 聚合酶链反应测定人支气管肺泡洗出液细胞肿瘤坏死因子αmRNA 含量的方法。自一重组质粒(pAW 108)体外转录出互补 RNA(cRNA),此cRNA 含两段肿瘤坏死因子α特异性引物序列。将 cRNA 用分光光度计定量后取一定量和提取自人支气管肺泡洗出液细胞的 mRNA 混在一起做逆转录,得到的 cDNA 混合物经1:3稀释后以肿瘤坏死因子α特异性引物做 DNA 聚合酶链反应放大.在反应中加入一定量末端标记^(32)P 的引物.放大产物经琼脂糖凝胶电泳分带,将各区带切下做β-液闪计数.以各区带的放射性计数对 cRNA 含量和细胞数做图,根据已知的 cRNA 的含量从图上可以推出一定量的细胞中的肿瘤坏死因子αmRNA 的含量。
TNFα mRNA in human bronchoalveolar lavage(BAL)cells was quantitated by a olymerase chain reaction(PCR)using a cRNA internal standard,cRNA molecules were in vitro transcribed from pAW108 plasmid in which the sequences of upstream primer and omolementary sequences of downstream primer of TNFα were inserted.The cRNA and mRNA extracted from BAL cells were mixed and reverse-transcribed into cDNA.The resultant cDNA mixture was 1:3 diluted and amplified by PCR using TNFα specific primers.^(32)p-labeled upstream primers were included in the PCR reaction,cDNA fragments amplified was run on a 3% agarose gel.The radioactivity of positive bands was determined in a scintillation ounter.After plotting variable template concentrations of the internal standard pAW108 cRNA and the number of BAL cells against the radioactivity of their PCR products,the levels of TNFα mRNA in BAL cells were quantitated by comparision to those of cRNA internal standard.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1992年第2期100-104,共5页
Chinese Journal of Immunology
基金
香港郑裕同先生基金
关键词
mRNA定量
肿瘤坏死因子Α
BAL
Quantitation of mRNA
Polymerase chain reaction
Tumor necrosis factor
