Hybridisation buffer

The conventional microarray hybridisation was performed in gasket hybridisation chambers of a volume of 100 μl each (Agilent, Santa Clara, CA, USA). The hybridisation mixture, comprising 20 μL of the fluorescence labelled amplification product, 40 μL hybridisation buffer (Roche Diagnostics GmbH, Rotkreuz, Switzerland), and 40 µL ddH₂O, was applied to the hybridisation chambers. The reaction occurred in a hybridisation oven (Microarray Hybridisation Chambers, Agilent, Santa Clara, CA, USA) at 55 °C for 1 h. The slides were subsequently washed repeatedly, first with 2 × SSC buffer containing 0.1% sodium dodecyl sulphate (SDS) for 5 min, second with 0.2 × SSC buffer for 2 min, and third with ddH₂O for 1 min. The slides were dried by centrifuging.

The sections (4 μm) of cervical cancer tissues and adjacent normal cervical tissues were treated with proteinase K (20 mg/mL) for 15 min and refixed in 4% PFA for 15 min. After acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, sections were prehybridised with hybridisation buffer (Roche, Mannheim, Germany) at 40 °C for 2 h and then hybridised with a DIG-labelled LNA-miR-205 probe (5′-CAG(+A)C(+T)CCGG(+T)GGAA(+T)GA(+A)GGA-DIG-3′) at 40°Covernight. The sections were then incubated in buffer containing anti-DIG-antibody (Roche) 2 h at 37 °C, followed by staining with NBT and BCIP (Promega, Madison, WI, USA). Samples were viewed under a Nikon TE 2000-U microscope (Nikon, Tokyo, Japan).