Expression data from NIH-3T3 cells used for half-life determination

Data from tc-, nt- and p-RNA as well as 1 and 2h of actinomycin-D treatment (5µg/ml) of NIH-3T3 cells used to determine half-lives. RNA was labeled for 15, 30 or 60 minutes with 4-thiouridine. After preparation of tc-RNA, thiol-labeled RNA was biotinylated using biot-HPDP and subsequently tc-RNA was separated into nt- and p-RNA using streptavidin coated magnetic beads. All three fractions were used for microarray analysis. For actinomycin-D experiments only tc-RNA was used prepared from cell before and 1 an 2h after addition of act-D. We used microarrays to analyze the effects of 1 and 3h of IFNalpha and gamma treatment in total cellular RNA Keywords: determination of RNA half-lives in NIH-3T3 NIH-3T3 cells ( 5th to 15th passage after thawing) were split from confluent plates 24h before start of the experiment. At the begin of the experiment about 80% confluency was reached. The experiment was started by applying fresh, prewarmed, CO2-equilibrated medium containing either 200µM 4sU (for 1h labeling), 500µM 4sU (for <=30min labeling) or 5µg/ml actinomycin-D.