In vitro synthesis of capped transcripts

Capped RNA transcripts containing modified cap analogs were synthesized by in vitro transcription of plasmid pluc-A60 digested with NcoI using T7 RNA polymerase, which yielded a capped RNA corresponding to the first 48-nt of firefly luciferase mRNA. All transcripts were obtained in the presence of given cap analogs and 10 µCi/µL [α-³²P] GTP (PerkinElmer) as described previously (Su et al. 2011). Reaction mixtures were extracted with phenol and chloroform; RNAs were separated from small-molecule impurities with NucAway spin columns (Ambion). The concentrations of RNAs were determined via measuring Cerenkov radiation in a scintillation counter (Beckman).