Northern Blot Analysis of Nluc mRNA

For northern blot experiments, cells were transfected in six-well format with the indicated reporter gene, Nluc 4xMS2 pA. Purified total RNA (3 µg) was separated by formaldehyde-agarose gel electrophoresis with 1x MOPS buffer and then transferred to Immobilon NY+ member (Millipore) as previously described (Arvola et al. 2020 (link)). The RNA integrity and loading of the RNA in each sample was assessed by staining with ethidium bromide. The blot was prehybridized for 1 h at 68°C in 10 mL of UltraHyb buffer (Invitrogen) and then was probed with an antisense RNA Nluc probe that was transcribed with α-³²P UTP (PerkinElmer) as described (Arvola et al. 2020 (link)). After overnight hybridization, the blots were washed twice in 25 mL of 2x SSC, 0.1% SDS and then twice in 0.1x SSC, 0.1% SDS at 68°C for 15 min. Blots were visualized by phosphorimaging with a Typhoon FLA phosphorimager (GE Life Sciences) and quantified using ImageQuant TL software (GE Life Sciences). The 18S rRNA was detected using a 5′ ³²P-labeled antisense oligodeoxynucleotide probe (listed in Supplemental Table S4), prepared as previously described (Arvola et al. 2020 (link)). Prehybridized was done in 10 mL UltraHyb buffer for 1 h at 42°C and then the probe was incubated with the blot overnight. Blots were then washed twice with 25 mL 2×SSC containing 0.5% SDS for 30 min each wash at 42°C.