Northernmax formaldehyde loading dye

MA-104 cell monolayers in 6 well plates were either mock infected or infected with SHFV infectious clone virus at a MOI of 1. At different times after infection, total cell RNA was extracted with TRI reagent (Molecular Research Center, Inc.). One μg of RNA was mixed with NorthernMax formaldehyde loading dye (Ambion), denatured at 80°C for 10 min and then separated on a 1% formaldehyde agarose gel for 2.5 h at 100V. RNA markers (Millennium Markers-Formamide, Ambion) were run on one lane of the gel. RNA was transferred overnight by capillary action onto an Amersham Hybond-N⁺ membrane (GE Healthcare) and the RNA was then UV-crosslinked to the membrane. The lane containing the RNA standards was cut from the membrane and stained with methylene blue. The rest of the membrane was pre-hybridized with DIG Easy Hyb buffer (Roche) at 68°C for 30 min and then hybridized with 100 ng/mL of a DIG-labeled, denatured RNA probe at 68°C overnight. The hybridized membrane was washed first with low stringency buffer (2X SSC + 0.1% SDS) at room temperature, then with high stringency buffer (0.1X SSC + 0.1% SDS) at 68°C followed by blocking with DIG blocking solution (Roche). To detect the RNA bands, membranes were incubated with anti-DIG antibody (1:10,000 dilution) (Roche), developed with CDP-Star (Roche) and imaged with an LAS4000 mini Luminescent Image Analyzer (GE Healthcare).