Saed G M, Fivenson D P
Department of Dermatology, Henry Ford Hospital, Detroit, MI.
Biochem Biophys Res Commun. 1994 Sep 15;203(2):935-42. doi: 10.1006/bbrc.1994.2272.
We have developed a method to accurately quantitate IFN gamma mRNA in HUT-78 cells before and after PUVA treatment by the competitive RT/PCR technique, which could be utilized to accurately quantitate any mRNA species of interest. Total RNA was isolated from HUT-78 cells before and after PUVA treatment. A synthetic IFN gamma mRNA was made to contain a 54 bp deletion in the middle of IFN gamma cDNA gene and used as an internal standard. 0.5 microgram of target RNA was co-reverse transcribed and co-amplified with increasing concentrations of synthetic IFN gamma RNA using the same primers. The products of the synthetic RNA were separated from that of the target RNA by gel electrophoresis. This allowed determination of the amount of target IFN gamma mRNA to be quantitated by extrapolating against a standard curve. PUVA treatment of HUT-78 cells resulted in an increase in IFN gamma mRNA level from 32 to 80 pg/microgram of total RNA, suggesting that PUVA induces transcription of T-helper 1 cytokines as part of its mechanism of action.
我们已经开发出一种方法，通过竞争性逆转录/聚合酶链反应（RT/PCR）技术，准确测定补骨脂素联合紫外线A（PUVA）处理前后HUT-78细胞中γ干扰素（IFNγ）信使核糖核酸（mRNA）的含量，该方法可用于准确测定任何感兴趣的mRNA种类。从PUVA处理前后的HUT-78细胞中分离出总RNA。制备一种合成的IFNγ mRNA，使其在IFNγ互补DNA（cDNA）基因中间有一个54碱基对的缺失，并用作内标。使用相同引物，将0.5微克靶RNA与浓度不断增加的合成IFNγ RNA共同逆转录和共同扩增。通过凝胶电泳将合成RNA的产物与靶RNA的产物分离。这样就可以通过根据标准曲线外推来确定待定量的靶IFNγ mRNA的量。PUVA处理HUT-78细胞导致IFNγ mRNA水平从每微克总RNA 32皮克增加到80皮克，这表明PUVA诱导辅助性T细胞1细胞因子的转录是其作用机制的一部分。
