Northern Blotting.

RNA probe complementary to a fragment (∼350 nt) of RLuc sequence was in vitro transcribed by using a HiScribe T7 high-yield RNA synthesis kit (NEB) with DIG-11-UTP (Roche) and 200 ng of template DNA (PCR product), and purified by using Monarch RNA Cleanup Kit (NEB). Total RNA was extracted from HEK293T cells using TRIzol (Invitrogen) and treated with TurboDNase at 37 °C for 30 min. For each sample, 5 µg of total RNA was mixed with 5× RNA loading buffer (2.5 mg/mL bromophenol blue, 12 mM EDTA, 2.76% formaldehyde, 20% glycerol, 30.84% formamide, 80 mM MOPS, 20 mM NaOAc), denatured at 65 °C for 10 min, rapidly cooled on ice, and loaded on a 1% formaldehyde agarose gel (1% agarose, 0.67% formaldehyde, 20 mM MOPS, 5 mM NaAc, 2 mM EDTA, pH 7.0). Formaldehyde agarose gels were run at 5 V/cm in 1× running buffer (0.74% formaldehyde, 20 mM MOPS, 5 mM NaOAc, 2 mM EDTA, pH 7.0), stained with SYBR Gold (Thermo Fisher) in 1× TBE buffer, and imaged using a Gel Doc XR system (Bio-Rad). RNA was transferred to a BrightStar-Plus positively charged nylon membrane (Thermo Fisher) by overnight capillary transfer in 20× SSC and cross-linked by 254-nm UV. After prehybridization, membranes were incubated with 100 ng/mL denatured DIG-labeled RNA probe in DIG EasyHyb buffer (Roche) at 68 °C overnight, washed twice in 2× SSC, 0.1% SDS at room temperature, twice in 0.1× SSC, 0.1% SDS at 68 °C, and incubated with anti-DIG-alkaline phosphatase conjugate (Roche). Chemiluminescent signals were developed using CDP-Star (Roche) according to the manufacturer’s instruction and detected by using an Odyssey CLx system (Li-Cor).