Figure 1.

Procedure used to label newly synthesized RNA molecules with 4-TU. (A) Flow chart and (B) scheme showing the labeling of new RNAs with 4-TU, followed by derivatization with biotin and purification of tagged RNAs. After reverse transcription, any individual cDNA (RNA) species can be quantitated by qPCR. (C and D) HeLa cells were pre-cultured with actinomycin D for 30 min at different concentrations, as indicated (high: 2 μg/ml or low: 20 ng/ml), and then the cells were incubated with 4-TU 100 nM for 3 h. 4-TU-labeled RNAs were extracted and purified as described in the ‘Materials and Methods’ section. The amounts of cDNAs derived from specific RNAs were quantitated by RT–qPCR and are expressed as described in the ‘Materials and Methods’ section: (C) Data for 18S rRNA (also measures its precursors); (B) Data for 5S rRNA.