Clinical Research Directory

Gene expression was done by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Paraffin-embedded biopsies were sectioned into 10-μm-thick slices, two of which were deposited into 1.5-ml microcentrifuge tubes and dewaxed via immersion in xylene at 50 °C, followed by absolute and 96% ethanol. Extraction of total RNA (including miRNA) was performed using the Qiagen RNeasyPlus Universal Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. Briefly, the samples were homogenized in QIAzol Lysis Reagent using TissueRuptor. RNA was further precipitated with chloroform, mixed with 1.5 volumes of 100% of Ethanol and following precipitation and washing steps eluted in RNase-free water. The concentration of extracted RNA was assessed using UV-spectrophotometry. MiRNA expression was quantitatively evaluated using either the TaqMan miRNA assay (Applied Biosystems, CA, USA) or SYBR Green (RNU6b) method. Approximately 20 ng of total RNA were reverse tran