CLINICS 2012;67(3):255-259 BASIC RESEARCH DOI:10.6061/clinics/2012(03)09 Isolated total RNA and protein are preserved after thawing for more than twenty-four hours Ivone Braga de Oliveira, De´ bora Rothstein Ramos, Karen Lucasechi Lopes, Regiane Machado de Souza, Joel Claudio Heimann, Luzia Naoˆ ko Shinohara Furukawa Faculdade de Medicina da Universidade de S a˜o Paulo, Department of Internal Medicine, Laboratory of Experimental Hypertension, S a˜o Paulo/SP, Brazil. OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of b-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues. KEYWORDS: Thawed tissue; Defrosted isolated total RNA; Ultra-low temperature; Molecular viability; Western blot. Oliveira IB, Ramos DR, Lopes KL, Souza RM, Heimann JC, Furukawa LN. Isolated total RNA and protein are preserved after thawing for more than twenty-four hours. Clinics. 2012;67(3):255-259. Received for publication on September 27, 2011; First review completed on October 3, 2011; Accepted for publication on October 25, 2011 E-mail: luzia@usp.br Tel.: 55 11 3061-8362 INTRODUCTION It is commonly recommended to store samples of plasma, tissues, and isolated total RNA in an ultra-low-temperature freezer to preserve the tissue structure and biochemical parameters by decreasing or inhibiting enzyme activity and other processes. Due to a technical problem, an ultra-low-temperature freezer in our laboratory defrosted, and the samples were kept at room temperature for more than twenty-four hours. Therefore, we decided to determine if the samples could still be used for some analyses. Kisand et al. (1) and Yokomuro et al. (2) observed that some but not all blood biomarkers are preserved after freeze/thaw cycles. In other studies, it was observed that freeze-thaw cycles induce biomechanical changes in arteries (3) and render sciatic nerve tissue inviable for grafting (4). Molecular analyses using cancer tissues have shown that storing the tissue or performing Copyright 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. 255 the fixation with formalin at a low temperature can prevent the degradation of nucleic acids (5,6). However, no information was found in the literature with regard to whether material that has experienced a pro- longed thaw could be useful for either biochemical or molecular determinations. Because ultra-low-temperature freezers may defrost due to electrical or other problems, it is important to determine whether thawed biological material retains sufficient integrity for certain analyses. Therefore, the present study was designed to evaluate samples that were defrosted for more than twenty-four hours with regard to the mRNA and protein levels, the histology of intact tissue and the integrity of isolated total RNA. MATERIALS AND METHODS Samples The studied samples were thawed due an eletric problem in ultra-low freezer (-80˚C) and remained at room tempera- ture for at least twenty-four hours. After that, they were transferred back to an ultra-low freezer (-80˚C). Control, unthawed samples were also evaluated. The materials listed below were studied. Thawed tissues: heart (n = 12), pancreas (n = 6), brown adipose tissue (BAT, n = 9), white adipose tissue (n = 9), liver