Zeitschrift für Molekulare Histologie und Medizinische Physiologie

Increased Detection of RNA Species in Histological Tissues Using a Two-Temperature Fixation Protocol

Abstract

Abbey P. Theiss, David Jiang, Daniel R. Bauer, Anne Pedata, Dipti Tillu, Mark D. Robida, Michael Otter, William A. Day and David R. Chafin

A number of diagnostic and research assays rely on accurately measuring the levels of ribonucleic acids (RNAs) in tissue. The best way to obtain high quality intact RNA from tissues is to use samples that are fresh. The standard for treating tissues used in pathology workflow is to treat with 10% neutral buffered formalin, alcohols, xylenes and waxes but produces lower quality material for detection of RNA. One barrier to analyzing RNA species in formalinfixed paraffin embedded tissues (FFPE) is the use of variable times that institutions fix tissue samples which causes variable preservation and assay results. Another barrier is how long samples sit at room temperature (RT) before fixing, so called cold ischemia time. We report here on increased detection of RNA species from our rapid twotemperature formalin fixation protocol (cold+warm) which standardizes tissue collection and reduces cold ischemic time as variables in RNA ISH analysis. Human tonsils, mouse xenograft tumors and cell culture samples were used as model systems in fixation time course experiments to determine sensitivity of RNA preservation to the level of fixation. To extend these results colon, breast and lung carcinoma clinical patient samples were also examined. The amount of detectable β-actin RNA or miRNAs increased with increasing fixation times in model systems. In clinical tissues, the amount of detectable RNA species dramatically increased (20-40 fold) in tissues fixed with the cold +warm method. A dramatic reduction (20-50 fold) in RNA levels was observed when the tissues had longer cold ischemia times with standard room temperature fixation.