Mini quick g 25

Radiolabeled probes were prepared in 20 μL reactions containing 10 pmoles of antisense oligonucleotide (ASO) to HSUR 2, U2, U6, miR-142-3p, miR-16 or miR-21, 10 units of T4 polynucleotide kinase (New England Biolabs) and 151.5 μCi of [γ-³²P]ATP (6000 Ci/mmol, PerkinElmer) and incubated at 37°C for one hour. Unincorporated isotope was removed by centrifugation using Mini Quick® G-25 gel filtration columns following the manufacture’s protocol (Roche). Radiolabeled probes were eluted in a volume of 50 μL of H₂O and 10 μL were used in each hybridization experiment. A β-actin probe was labeled using 50 μCi of [α-³²P]dCTP and Klenow DNA polymerase for 15 min using the DecaPrime II kit according to the manufacturer’s instructions (Ambion). A 10-μL aliquot of the radiolabeled probe was taken to a volume of 100 μL with 10 mM EDTA, pH 8.0 and incubated at 95°C for 10 min. The labeled probe was then added directly without further purification to the prehybridized membranes.