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.