Wundrack I, Dooley S
Institute of Human Genetics, Saar University, Homburg, Germany.
Electrophoresis. 1992 Sep-Oct;13(9-10):637-8. doi: 10.1002/elps.11501301130.
A sensitive nonradioactive ribunuclease protection assay is described which we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)-rUTP instead of [alpha-32P]CTP or [alpha-32P]UTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.
本文描述了一种灵敏的非放射性核糖核酸酶保护分析方法，我们已用此方法研究GLC4（一种基因扩增的人小细胞肺癌细胞系）中c-myc基因的转录及启动子使用情况。对于体外转录，我们使用地高辛（DIG）-rUTP代替[α-32P]CTP或[α-32P]UTP，聚丙烯酰胺凝胶电泳后，使用电转印法将受保护的探针转移至来自宝灵曼公司的尼龙膜上。随后，通过化学发光检测对膜进行分析。曝光1小时后获得结果，这些结果与使用放射性检测得到的结果相当。
