Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection.
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.
Wundrack
I
I
Institute of Human Genetics, Saar University, Homburg, Germany.
Dooley
S
S
D018288:Carcinoma, Small Cell / Q000235:genetics; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D016259:Genes, myc; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008175:Lung Neoplasms / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012260:Ribonucleases; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D012333:RNA, Messenger; C070202:digoxigenin-11-deoxyuridine triphosphate; D012260:Ribonucleases; D004076:Digoxigenin