Jan Schmeller1, Michael Wessolly2, Elena Mairinger2, Sabrina Borchert2, Thomas Hager2, Thomas Mairinger3, Kurt Werner Schmid2, Jeremias Wohlschlaeger2, Robert F H Walter4, Fabian D Mairinger2. 1. Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. Electronic address: jan.schmeller@uk-essen.de. 2. Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. 3. Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany. 4. Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; Ruhrlandklinik, West German Lung Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Abstract
INTRODUCTION: The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis. DESIGN: FFPE-derived RNA from sections of 23 patients with spontaneous pneumothoraxes was used. Unstained sections of FFPE tissue were stored at various temperatures (-80 °C, -20 °C, 4 °C, 24 °C) prior to RNA extraction. The potential impact on RNA quality of semi-automatic and manual RNA isolation and three different deparaffinization agents (mineral oil, xylene and d-limonene) were compared. RESULTS: The storage temperature of FFPE sections affects RNA concentration and fragmentation, with the optimal storage temperature below -20 °C. The RNA extracted with d-limonene shows equivalent quality to the RNA extracted using more toxic standard agents. The manual isolation provides a higher RNA yield compared to the semi-automatic isolation. However, no differences in the amount of longer RNA fragments were observed. Furthermore, the semi-automatic isolation showed an enhanced RNA quality. CONCLUSION: FFPE sections not directly used for RNA extraction should be stored below -20 °C to increase quality and yield of the RNA. Usage of semi-automatic isolation produces superior results and simplifies routine processes by having less hands-on-time. Replacement of toxic xylene by d-limonene may contribute to improved occupational safety while not influencing analytical results.
INTRODUCTION: The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis. DESIGN: FFPE-derived RNA from sections of 23 patients with spontaneous pneumothoraxes was used. Unstained sections of FFPE tissue were stored at various temperatures (-80 °C, -20 °C, 4 °C, 24 °C) prior to RNA extraction. The potential impact on RNA quality of semi-automatic and manual RNA isolation and three different deparaffinization agents (mineral oil, xylene and d-limonene) were compared. RESULTS: The storage temperature of FFPE sections affects RNA concentration and fragmentation, with the optimal storage temperature below -20 °C. The RNA extracted with d-limonene shows equivalent quality to the RNA extracted using more toxic standard agents. The manual isolation provides a higher RNA yield compared to the semi-automatic isolation. However, no differences in the amount of longer RNA fragments were observed. Furthermore, the semi-automatic isolation showed an enhanced RNA quality. CONCLUSION: FFPE sections not directly used for RNA extraction should be stored below -20 °C to increase quality and yield of the RNA. Usage of semi-automatic isolation produces superior results and simplifies routine processes by having less hands-on-time. Replacement of toxic xylene by d-limonene may contribute to improved occupational safety while not influencing analytical results.
Authors: Amelia Meecham; Elena Miranda; Hayley T Morris; Jane Hair; Karin A Oien; Gareth Gerrard; Naomi Guppy; David Mooney; Emily C Shaw; Margaret Ashton-Key; Robert Lees; Adrienne Flanagan; Manuel Rodriguez-Justo Journal: Histochem Cell Biol Date: 2021-12-14 Impact factor: 4.304