Defrosted isolated total RNA is preserved Olivera IB et al. Figure 1 - The A260/A280 ratios of the total RNA extracted from the thawed and unthawed tissue samples. (n = 7), and kidney (n = 12) obtained from twelve-week-old male Wistar rats. Unthawed tissue: liver (n = 7) and kidney (n = 3). Thawed (n = 18) and unthawed (n = 17) isolated total kidney RNA. Thawed tissue homogenate: kidney (n = 3), heart (n = 3), and BAT (n = 3). Unthawed tissue homogenate: kidney (n = 3). Total RNA extraction The total RNA was extracted from 50 mg of thawed samples of kidney (n = 3), liver (n = 4), heart (n = 5), pancreas (n = 3), white adipose tissue (n = 5), BAT (n = 3), and from unthawed samples (liver, n = 7) using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA, http:// www.invitrogen.com) according to the manufacturer’s instructions. The total RNA pellets were resuspended in 100 mL of diethyl pyrocarbonate-treated water and stored at -80˚C until use. The total RNA concentrations in thawed and unthawed tissue and in thawed and unthawed isolated total RNA samples were determined by measuring the absorbances at 260 nm and 280 nm, and the integrity was verified by ethidium bromide fluorescence. Using reverse transcriptase, cDNA was synthesized from 1 mg of thawed isolated total RNA (kidney, n = 18) or unthawed isolated total RNA (kidney, n = 17) using an oligonucleotide dT primer (Promega Corporation, Madison, WI, USA, http:// www.promega.com) and the ImProm II reverse transcriptase enzyme (Promega Corporation, Madison, WI, USA, http:// www.promega.com). CLINICS 2012;67(3):255-259 A polymerase chain reaction (PCR) was performed to amplify the following specific cDNAs: renin (sense, 59- CATTACCAGGGCAACTTTCAC-39, and antisense, 59- TCATCGTTCCTGAAGGAATTC-39) and b-actin (sense, 59- TATGCCAACACAGTGCTGTCTGG, and antisense, 59- TACTCCTGCTTGCTGATCCACAT-39). The sizes of the reaction products were estimated using a 100 bp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA, USA, http://www.invitrogen.com). The renin and b-actin mRNA levels were determined using the isolated thawed and unthawed total RNA samples. The PCR mixture contained 1 ml of cDNA, 2.5 mL of 10X buffer (100 mM Tris–HCl [pH 8.5] and 500 mM potassium chloride), 0.5 ml of 10 mM deoxynucleotide triphosphate mix, 0.75 ml of 50 mM magnesium chloride, 15 pM of each primer, 2 U of Taq DNA polymerase (LGC Biotecnologia, Cotia, SP, Brazil, http://www.lgcbio.com.br), and diethyl pyrocarbo- nate-treated water in a total volume of 25 ml. The cDNAs were amplified under the following conditions: 94.0˚C for 3 minutes; 35 cycles of 94.0˚C for 1 minute, 51.5˚C (for renin) or 57.8˚C (for b-actin) for 1 minute, and 72.0˚C for 1 minute; and 72.0˚C for 10 minutes. Negative controls were routinely performed by omitting the cDNA from the PCR mixture. The PCR products were electrophoresed on 2.0% agarose gels with ethidium bromide, and the bands were semi-quantified using image analysis software (Alpha ImagerTM 1220 version 5.5; Alpha Innotech Corporation, San Leandro, CA, USA, http://www.alphainnotech.com). All of the PCRs produced a single band of the predicted size. Protein levels The protein level was measured by Western blotting according to the following protocol. Samples of thawed kidney (n = 6), heart (n = 3), brown adipose tissue (BAT, n = 3), and unthawed kidney (n = 3) were homogenized separately in lysis buffer (10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesul- fonic acid [HEPES] [pH 7.6], 100 mmol/L potassium chloride, 3 mmol/L magnesium chloride, 5 mmol/L Ethylenediamine tetraacetic acid [EDTA], 10% glycerol, 1 mmol/L dithiothrei- tol, 10% sodium dodecyl sulfate [SDS], and 1:10 protease inhibitor cocktail [Sigma-Aldrich Corporate Offices, MO, Canada, http://www.sigmaaldrich.com]) and centrifuged at 10,000 x g. The supernatant was recovered, and the protein concentration was determined using the Bradford method (7). Aliquots of 100 mg of total protein from the homogenized thawed and unthawed tissue and thawed homogenate (kidney, n = 3; heart, n = 3; BAT, n = 3) were separated on a 12% SDS polyacrylamide gel. Figure 2 - The 28S and 18S bands of the isolated total RNA from the control (unthawed) and thawed tissues. 1 and 2 = control liver; 3 and 4 = liver; 5 and 6 = kidney; 7 and 8 = heart; 9 and 10 = white adipose tissue; 11 = pancreas; 12, 13 and 14 = brown adipose tissue; 15 = neonate heart. 256