Illustra g 25 microspin columns

20 μg of total RNA isolated with Trizol Reagent (Life Technologies) was separated on a 12% polyacrylamide/Urea/TBE gel and transferred to HyBond N+ membrane (GE Healthcare) using the TransBlot SD. Semi-dry Transfer System (Bio-Rad). DNA oligo probes were γ-³²P end labeled using T4 polynucleotide kinase (New England Biolabs) and purified using Illustra G-25 MicroSpin columns (GE Healthcare). Blots were blocked in UltraHyb Oligo (Life Technologies) at 42°C for 30 min and hybridized with 5′ labeled DNA probe overnight at 42°C. Blots were washed twice with pre-warmed 2xSSC and 0.5% SDS at 42°C for 30 minutes. RNA was visualized with phosphor screens from GE-HealthCare.

Total RNA was separated on a 12% polyacrylamide/urea/TBE gel (Sequagel, National Diagnostics) and transferred to a HyBond N⁺ membrane (GE Healthcare) using the TransBlot SD semidry transfer system (Bio-Rad). RNA was then UV cross-linked to the membrane. DNA oligo probes perfectly complementary to the miRNAs of interest were γ-³²P end-labeled using T4 polynucleotide kinase (New England Biolabs) and purified using Illustra G-25 MicroSpin columns (GE Healthcare). Membranes were prehybridized in UltraHyb oligo (Ambion) for at least 30 min at 42°C and then hybridized with a radiolabeled DNA probe overnight at 42°C. Blots were washed twice for 30 min with 2× SSC and 0.5% SDS prewarmed to 42°C. RNA was visualized by exposure to phosphor screens and then imaged on a Storm scanner (Molecular Dynamics).