Rna millenium marker

The northern blot protocol for longer RNA molecules (>200) was as follows: 3.5 μg total RNA from MHV68-infected or mock-infected murine fibroblasts was loaded onto 1% agarose gel containing 6% formaldehyde in parallel with RNA Millenium Marker (Ambion). The gel was run in MOPS buffer, then blotted onto an Hybond XL nylon membrane (Life Technologies) overnight with Turblotter kit in 20X SSC buffer. Following washing, the RNA was crosslinked to the membrane by UV, and the membrane was stained with 0.02% methylene blue for visualization of RNA integrity and markers. The membrane was then pre-hybridized at 63°C for 4 hours in ULTRAhyb (Ambion) buffer. The ³²P-labeled RNA probes were generated with Maxiscript T7/Sp6 Transcription Kit (Ambion) using PCR amplicons specific for ORF75A/B/C regions (S3 Table). Subsequently, RNA probes were added and incubated overnight. Following washes with 1x SSC buffer, the membrane was exposed to film at 80°C.

RNA extraction and northern blot protocol is mainly based on the protocol by McClure et al. [17 (link)]. Briefly, 2 × 10⁶ NIH 3T12 fibroblasts were infected at multiplicity of infection (MOI) 5 with MHV68, and at 24 hpi cells were harvested, lysed in Trizol, and RNA was extracted according to the manufacturer’s protocol [17 (link)]. The northern blot protocol for longer RNA molecules (>200) was as follows: 5 μg total RNA sample was loaded onto a 6% formaldehyde-containing 1% agarose gel RNA Millenium Marker (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). The gel was run in a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, then blotted onto a Hybond XL nylon membrane (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) overnight with Turbolotter kit in 20X SSC buffer. The membrane was washed, RNA was crosslinked to the membrane using UV light (Spectrolinker XL1500 UV Crosslinker, Spectronics Corporation, Westbury, NY, USA), and the membrane was stained with 0.02% methylene blue for visualization of the RNA integrity and markers. The crosslinked membrane was prehybridized at 63 °C for 4 hours in an ULTRAhyb (Ambion) buffer and then labeled probe (see below) was added for overnight incubation. The next day the membrane was washed three times with a 1x SSC buffer and exposed to a film at 80 °C for appropriate time.