Tissue Preparation for RNA and Histology

Organs were prepared from adult Wistar rats, cut to appr. 3–5 mm thick pieces, and immediately submerged in 10% neutral buffered formalin for fixation and embedding in paraffin, or stabilized in RNAlater solution (QIAGEN GmbH, Hilden, Germany), for reference samples with ideal RNA preservation, respectively. Formalin fixation of tissue specimens was performed over night (20–24 h) at room temperature, unless otherwise noted. Specimens were dehydrated in 70%, 90%, and 100% ethanol, followed by xylene, for 2×1 h each solution, then embedded in Paraplast Plus (Sherwood Medical Co., St. Louis) for 2–3 h at 60°C. For RNA isolation, 10 µm sections were cut on a standard microtome. In each case, the first 2 sections were discarded to exclude negative effects from exposure to air.
FFPE specimens from surgically removed lung carcinoma were obtained using routine diagnostic procedures; the study was approved by the ethical committee of the Ärztekammer Hamburg.