Ambion gel loading buffer 2

Northern blotting for miRNA detection was performed with minor modifications as described previously [4 (link)]. Briefly, 10 μg of RNA denatured in Ambion gel loading buffer II (Life technologies) at 75°C for 15 min was loaded and ran on a 15% TBE-Urea gels (Invitrogen, Carlsbad, CA, USA) at 180 V for 1 h. The gel was then transferred onto a nylon membrane using an iBLOT DNA transfer stack (Invitrogen) as per the manufacturer's instructions. Subsequently, the membrane was crosslinked at 1200 kJ using a STRATALINKER (Stratagene, La Jolla, CA, USA). The membrane was then prehybridized in the prehyb buffer (Sigma-Aldrich) at 37°C for 1 h followed by the addition of 1.2 pmol/mL of LNA-modified 5′ and 3′ DIG-labeled hsa-miR-1290 probe (Exiqon) to the buffer and hybridization overnight at 37°C. The next day, the membrane was washed for 5 min with a low stringency wash buffer (2 × SSC, 0.1% SDS) followed by 2 × washes for 20 min each with a high stringency wash buffer (0.5 × SSC, 0.1% SDS) and a final wash for 20 min with ultrahigh stringency wash buffer (0.1 × SSC, 0.1% SDS). After the washes, the membrane was blocked for 1 h with 1 × blocking buffer for 1 h followed by incubation with anti-DIG AP antibody in a 1:  20 000 dilution in blocking buffer. Finally, DIG signal development was carried out using the DIG wash and block buffer set.