Defrosted isolated total RNA is preserved Olivera IB et al. CLINICS 2012;67(3):255-259 Figure 5 - Protein level of b-actin in the thawed homogenates (samples: 1 = kidney, 2 = heart and 3 = BAT), thawed tissues (samples: 4 = brown adipose tissue; 5, 6 and 7 = kidney; 8 = brown adipose tissue) and unthawed tissues (samples: 9 = kidney). test with Welchs correction analysis. Values of p,0.05 were considered statistically significant. The statistical analysis was performed using GraphPad 4.00 for Windows (9). Ethics The experiments in this study were previously approved by the Ethics Committee of Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. RESULTS A260/A280 ratio and the integrity of the total RNA from thawed tissues The A260/A280 ratio of the total RNA isolated from the thawed tissues was not affected relative to that of the unthawed tissues (Figure 1). However, when verifying the integrity, the 28S and 18S bands were not observed, indicating that the total RNA was degraded (Figure 2). A260/A280 ratio, total RNA concentration, RNA integrity and mRNA levels in thawed isolated total RNA samples Although the concentrations were the same (Figure 3B), the A260/A280 ratio was higher (p,0.05) for the thawed isolated total RNA than for the unthawed isolated total RNA (Figure 3A). The integrity of the total RNA was evaluated by gel electrophoresis analysis of the 28S and 18S bands (Figure 4A), and this analysis confirmed the integrity of the isolated total RNA after defrosting. By RT-PCR, we observed that the levels of b-actin (Figure 4B) and renin (Figure 4C) mRNA were perfectly maintained in the thawed isolated total RNA. From these data, it could be concluded that thawed isolated total RNA can be analyzed and processed even after defrosting for up to 24 hours. Protein levels The b-actin protein levels in the tissue and homogenate were analyzed by Western blotting, and these proteins were found not to have been degraded due to the defrosting (Figure 5). Histology We found that the morphology of some of the thawed tissues was not preserved relative to that of the respective unthawed tissues (Figure 6). DISCUSSION Some interesting and new observations were made in the present study. The samples used to determine the amount of intact mRNA in previously isolated total RNA and the protein levels in thawed intact tissues and homogenates are well preserved even after more than twenty-four hours at room temperature. These results suggest that, in the event of a technical problem with deep freezers that leads to defrosting, some material can still be used for some determinations. However, the histological structure and RNA extracted from the thawed tissues were not preserved. Okamoto et al. (10) have found that, during tissue freezing and thawing cycles, both the formation of ice crystals and the osmotic effect contribute to RNA degradation. In another study, it was found that RNA and DNA were stable only up to five hours after the tissue was excised when the tissue was maintained at room temperature (5). The results from the present study confirm that the RNA is not viable in defrosted tissues. The rapid degradation of RNA in tissues maintained at room temperature or submitted to freezing and thawing cycles may be due to RNAase digestion. The preservation of the thawed previously isolated RNA found in the present study supports this possible mechanism because RNAase is Figure 6 - Microphotographs of thawed and unthawed tissue. (A) kidney, (B) thawed kidney, (C) heart, (D) thawed heart, (E) BAT, (F) thawed BAT, (G) liver, (H) thawed liver, (I) white adipose tissue, (J) thawed white adipose tissue (400x). 258