Measurement of gene-specific transcription by nuclease protection of pulse-labeled nuclear RNA.
A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.
Greene
J J
JJ
Department of Biology, Catholic University of America, Washington, DC 20064.
Pearson
S L
SL
D000199:Actins / Q000235:genetics*; D005354:Fibrosarcoma / Q000235:genetics; D016259:Genes, myc; D006801:Humans; D007942:Leukemia, Experimental / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015138:RNA, Nuclear / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D014158:Transcription, Genetic; D014316:Tritium; D014529:Uridine / Q000378:metabolism
D000199:Actins; D015138:RNA, Nuclear; D014316:Tritium; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014529:Uridine