Synthesizing and Purifying RNA Probes for EMSA Analysis

The RNA probes were synthesized with the Riboprobe System-T7 (Promega) using DNA primer sequences immediately downstream from a T7 promoter, as previously described (26 (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 probes were designed to be around 30 nucleotides considering the location of the ARE motif, except for the Tnf-α probe, which was 58 nucleotides long. The synthesized RNA probes were separated from the free nucleotides using Sephadex G50 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 (12 (link)). The amount of RNA probe used in each lane of the EMSA was calculated to be ∼10 femtomoles. The sequence of the RNA probes used are listed below. Note that the three Ikzf2 probes were previously used in (27 (link)) and the last two probes, Gm-csf and Tnf-α were used as a positive control: