Synthesis and Purification of RNA Probes

The RNA probes were synthesized with the Riboprobe System-T7 (Promega) using synthetic DNA sequences immediately downstream from a T7 promoter, as previously described (Milligan et al. 1987 (link)). The RNA probes were body-labeled during the transcription process, which was performed in the presence of [α-³²P] UTP (3000 Ci/mmol; PerkinElmer). The synthesized RNA probes were separated from the free nucleotides using Sephadex G25 columns (GE Healthcare Life Sciences) and subsequently electrophoresed on a 16% polyacrylamide urea gel. The probes were purified from excised gel fragments after detection by autoradiography, as previously described (Ball et al. 2014 (link)). The amount of RNA used in each lane of the EMSA was calculated to be ∼10 femtomoles. The new mouse probes differ slightly; specifically for ARE2197 it starts and ends 3 nt earlier than the one previously published (Ball et al. 2014 (link)). A list of the RNA probe sequences used and their respective ID names are provided in Table 1.