Biometra eco maxi system

Northern blotting was performed according to a previously published protocol (Miyakoshi et al., 2019 (link)). Briefly, total RNA was isolated using the TRIzol reagent (Invitrogen), treated with TURBO DNase (Invitrogen), and precipitated with cold ethanol. RNA was quantified using NanoDrop One (Invitrogen). Total RNA (5 µg) was separated by gel electrophoresis on 6% polyacrylamide/7 M urea gels in 1 × TBE buffer for 3 hr at 250 V using Biometra Eco‐Maxi system (Analytik‐Jena). DynaMarker RNA Low II ssRNA fragments (BioDynamics Laboratory) were used as a size marker. RNA was transferred from the gel onto Hybond‐XL nylon membrane (GE Healthcare) by electroblotting for 1 hr at 50 V using the same system. The membrane was crosslinked with transferred RNA by 120 mJ/cm² UV light, incubated for prehybridization in Rapid‐Hyb buffer (Amersham) at 42℃ for 1 hr, and then incubated for hybridization with a [³²P]‐labeled probe JVO‐0749 and JVO‐0322 at 42℃ overnight to detect GcvB and 5S rRNA, respectively. The membrane was washed in three 15‐min steps in 5× SSC/0.1% SDS, 1× SSC/0.1% SDS, and 0.5× SSC/0.1% SDS buffers at 42℃. Signals were visualized on Typhoon FLA7000 scanner (GE Healthcare) and quantified using Image Quant TL software (GE Healthcare).