CLINICS 2012;67(3):255-259 absent from these samples. Therefore, isolated total RNA is protected from degradation due to thawing, maintaining the integrity of expression analyses. Accordingly, it is recom- mended that total RNA be isolated as soon as possible after tissue collection. The proteins in intact tissues are most likely denatured due to prolonged storage at room temperature. However, the protein level was preserved in the thawed intact tissues and homogenates. A possible explanation for this observation is that protein denaturation is inherent in the Western blot technique. In conclusion, both gene and protein expression analyses may be performed using total isolated RNA and intact tissue or homogenates, respectively, that have been stored for many hours at room temperature. AUTHOR CONTRIBUTIONS Oliveira IB performed the Western blotting and histology analysis and wrote the manuscript. Ramos DR, Lopes KL, and Souza RM performed the PCR-RT analysis and helped to write the manuscript. Heimann JC revised the manuscript. Furukawa LN conceived and coordinated the study and wrote the manuscript. REFERENCES 1. Kisand K, Kerna I, Kumm J, Jonsson H, Tamm A. Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Defrosted isolated total RNA is preserved Olivera IB et al. biobank samples. Clin Chem Lab Med. 2010;49:229-35, http:// dx.doi.org/10.1515/CCLM.2011.049. 2. Yokomuro H, Shiono N, Ozawa T, Fujii T, Watanabe Y, Koyama N, et al. Effect of cryopreservation on cell proliferation and immunogenicity of transplanted human heart cells. Ann Thorac Cardiovasc Surg. 2010; 16:105-12. 3. Venkatasubramanian RT, Wolkers WF, Shenoi MM, Barocas VH, Lafontaine D, Soule CL, et al. Freeze-thaw induced biomechanical changes in arteries: role of collagen matrix and smooth muscle cells. Ann Biomed Eng. 2010;38:694-706, http://dx.doi.org/10.1007/s10439-010- 9921-9. 4. Evans PJ, Mackinnon SE, Levi ADO, Wade JA, Hunter DA, Nakao Y, et al. Cold Preserved Nerve Allografts: Changes in basement membrane, viability, immunogenicity, and regeneration. Muscle Nerve. 1998; 21:1507-22, http://dx.doi.org/10.1002/(SICI)1097-4598(199811)21:11 ,1507::AID-MUS21.3.0.CO;2-W. 5. Jewell SD, Srinivasan M, McCart LM, Williams N, Grizzle WH, LiVolsi V, et al. Analysis of the molecular quality of human tissues. Am J Clin Pathol. 2002;118:733-41, http://dx.doi.org/10.1309/VPQL-RT21-X7YH- XDXK. 6. Bussolati G, Annaratone L, Medico E, DArmento G, Sapino A. Formalin fixation at low temperature better preserves nucleic acid integrity. PLoS ONE. 2011;6:e21043, http://dx.doi.org/10.1371/journal.pone.0021043. 7. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Clin Biochem. 1976;72:248-54, http://dx.doi.org/10.1016/ 0003-2697(76)90527-3. 8. Junqueira LC, Carneiro J. Histologia Ba´sica, 11th ed, Guanabara Koogan. 2008. 9. GraphPad Prism Software. Version 4.03. 2005. [Computer Program]. San Diego, CA, USA: GraphPad Inc. http://www.graphpad.com. 10. Okamoto M, Nakagata N, Toyoda Y. Cryopreservation and transport of mouse spermatozoa at – 79˚C. Exp Anim. 2001;50(1):83-86, http:// dx.doi.org/10.1538/expanim.50.83. 259