Detecting RNA Targets via Northern Blot

Northern blot analyses were performed using near infrared dye-labeled probes as previously described (35 (link), 36 (link)). Briefly, 15 µg of total RNA from either UM-SCC-10A or UM-SCC-12 was separated using 15% Urea-PAGE and contents were subsequently transferred to Hybond N+ membrane (GE) using LifeTech transfer module at 0.2 Amp for one hour. The membrane was crosslinked twice using 254nm UV crosslinker at 120 mJ/cm². The membrane was then placed in a hybridization oven and incubated with 10ml ExpressHyb hybridization solution (Takara) in a hybridization tube for 30 minutes at 30°C. IR-dye labeled probes and the membrane were then hybridized overnight at 30°C. After overnight hybridization, the membrane was washed twice, with 2x SSC buffer containing 0.1% SDS and 1x SSC buffer containing 0.1% SDS, respectively. For both washes, membrane was shaken at 110 rpm for 10 minutes at room temperature. Following washes, membrane was scanned on Amershan Typhoon scanner (GE health) to detect emission at 600 nm and 800 nm.