Dig easy hyb granule buffer

Total RNA was extracted from tissues using an miRNeasy Kit (Qiagen) according to the manufacturer’s instructions. Samples of 20 mg of total RNA were resolved using a 15% polyacrylamide gel containing 8 M urea and transferred to Hybond-N + nylon membrane (GE). After cross-linking with UV light, the membrane was pre-hybridized in DIG Easy Hyb granule buffer (Roche, Switzerland) for 30 min. Subsequently, the membrane was hybridized with a digoxigenin (DIG)-labelled DNA probe complementary to a specific miRNA sequence for 12 h at 40 °C. Signal detection was performed as described in the manual for a DIG High Prime DNA labelling and detection starter kit II (Roche, Switzerland).

Total RNAs, extracted from shrimp with the mirVana™ miRNA isolation kit (Ambion, Austin, TX, USA), were separated on a denaturing 15% polyacrylamide gel with 8 M urea. Then the RNAs were transferred to a Hybond-N+ nylon membrane (Amersham Biosciences, GE Healthcare, UK), followed by UV cross-linking. Then the membrane was prehybridized in 10 ml DIG Easy Hyb granule buffer (Roche, Basel, Switzerland) for 30 min and hybridized with digoxigenin (DIG)-labeled probe completely complementary to mja-miR-35 (5′ DIG-ACCGGAACTTCTCACAGTT-3′) overnight at 42°C. The DIG-labeled U6 probe (5′ DIG-GGGCCATGCTAATCTT CTCTGTATCGTT-3′) was used as a loading control. The membrane was incubated with an AP (alkaline phosphatase)-conjugated anti-DIG IgG (Roche) for 2 h at room temperature. Immunological detection was performed using the DIG High Prime DNA labeling and detection starter kit II (Roche).