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 &#946;-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

Furukawa, Luzia Naôko Shinohara

Heimann, Joel Claudio

Lopes, Karen Lucasechi

Oliveira, Ivone Braga de

Ramos, Débora Rothstein

Souza, Regiane Machado de

English

Cadernos Espinosanos (E-Journal)

BASIC RESEARCHIsolated total RNA and protein are preserved afterthawing for more than twenty-four hoursIvone Braga de Oliveira, De´bora Rothstein Ramos, Karen Lucasechi Lopes, Regiane Machado de Souza, JoelClaudio Heimann, Luzia Naoˆko Shinohara FurukawaFaculdade de Medicina da Universidade de Sa˜o Paulo, Department of Internal Medicine, Laboratory of Experimental Hypertension, Sa˜o Paulo/SP, Brazil.OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/orhistological 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 totalRNA 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 RNAconcentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chainreaction, 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 andlevel of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of b-actinprotein 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 isrecommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level ispreserved 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 thantwenty-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, 2011E-mail: luzia@usp.brTel.: 55 11 3061-8362INTRODUCTIONIt is commonly recommended to store samples of plasma,tissues, and isolated total RNA in an ultra-low-temperaturefreezer to preserve the tissue structure and biochemicalparameters by decreasing or inhibiting enzyme activity andother processes.Due to a technical problem, an ultra-low-temperaturefreezer in our laboratory defrosted, and the samples werekept at room temperature for more than twenty-four hours.Therefore, we decided to determine if the samples could stillbe used for some analyses.Kisand et al. (1) and Yokomuro et al. (2) observed that somebut not all blood biomarkers are preserved after freeze/thawcycles. In other studies, it was observed that freeze-thaw cyclesinduce biomechanical changes in arteries (3) and render sciaticnerve tissue inviable for grafting (4). Molecular analyses usingcancer tissues have shown that storing the tissue or performingthe fixation with formalin at a low temperature can prevent thedegradation of nucleic acids (5,6).However, no information was found in the literature withregard to whether material that has experienced a pro-longed thaw could be useful for either biochemical ormolecular determinations. Because ultra-low-temperaturefreezers may defrost due to electrical or other problems, it isimportant to determine whether thawed biological materialretains sufficient integrity for certain analyses.Therefore, the present study was designed to evaluatesamples that were defrosted for more than twenty-four hourswith regard to the mRNA and protein levels, the histology ofintact tissue and the integrity of isolated total RNA.MATERIALS AND METHODSSamplesThe studied samples were thawed due an eletric problemin ultra-low freezer (-80 C˚) and remained at room tempera-ture for at least twenty-four hours. After that, they weretransferred back to an ultra-low freezer (-80 C˚). Control,unthawed samples were also evaluated. The materials listedbelow were studied.Thawed tissues: heart (n = 12), pancreas (n = 6), brownadipose tissue (BAT, n = 9), white adipose tissue (n = 9), liverCopyright  2012 CLINICS – This is an Open Access article distributed underthe 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 theoriginal work is properly cited.No potential conflict of interest was reported.CLINICS 2012;67(3):255-259 DOI:10.6061/clinics/2012(03)09255(n = 7), and kidney (n= 12) obtained from twelve-week-oldmale Wistar rats.Unthawed tissue: liver (n = 7) and kidney (n= 3).Thawed (n = 18) and unthawed (n = 17) isolated totalkidney RNA.Thawed tissue homogenate: kidney (n= 3), heart (n = 3),and BAT (n = 3).Unthawed tissue homogenate: kidney (n= 3).Total RNA extractionThe total RNA was extracted from 50 mg of thawedsamples of kidney (n = 3), liver (n = 4), heart (n = 5), pancreas(n = 3), white adipose tissue (n= 5), BAT (n= 3), and fromunthawed samples (liver, n = 7) using the TRIzol reagent(Invitrogen Life Technologies, Carlsbad, CA, USA, http://www.invitrogen.com) according to the manufacturer’sinstructions. The total RNA pellets were resuspended in100 mL of diethyl pyrocarbonate-treated water and stored at-80 C˚ until use.The total RNA concentrations in thawed and unthawedtissue and in thawed and unthawed isolated total RNAsamples were determined by measuring the absorbances at260 nm and 280 nm, and the integrity was verified byethidium bromide fluorescence.Using reverse transcriptase, cDNA was synthesized from1 mg of thawed isolated total RNA (kidney, n=18) or unthawedisolated total RNA (kidney, n=17) using an oligonucleotide dTprimer (Promega Corporation, Madison, WI, USA, http://www.promega.com) and the ImProm II reverse transcriptaseenzyme (Promega Corporation, Madison, WI, USA, http://www.promega.com).A polymerase chain reaction (PCR) was performed toamplify 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 thereaction products were estimated using a 100 bp DNAladder (Invitrogen Life Technologies, Carlsbad, CA, USA,http://www.invitrogen.com).The renin and b-actinmRNA levels were determined usingthe isolated thawed and unthawed total RNA samples.The PCR mixture contained 1 ml of cDNA, 2.5 mL of 10Xbuffer (100 mM Tris–HCl [pH 8.5] and 500 mM potassiumchloride), 0.5 ml of 10 mM deoxynucleotide triphosphate mix,0.75 ml of 50 mMmagnesium 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-treatedwater in a total volume of 25 ml. The cDNAswereamplified under the following conditions: 94.0 C˚ for 3minutes; 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 routinelyperformed by omitting the cDNA from the PCR mixture. ThePCR products were electrophoresed on 2.0% agarose gelswith ethidium bromide, and the bands were semi-quantifiedusing image analysis software (Alpha ImagerTM 1220 version5.5; Alpha Innotech Corporation, San Leandro, CA, USA,http://www.alphainnotech.com). All of the PCRs produced asingle band of the predicted size.Protein levelsThe protein level was measured by Western blottingaccording to the following protocol. Samples of thawed kidney(n=6), heart (n=3), brown adipose tissue (BAT, n=3), andunthawed kidney (n=3) were homogenized separately in lysisbuffer (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 Ethylenediaminetetraacetic acid [EDTA], 10% glycerol, 1 mmol/L dithiothrei-tol, 10% sodium dodecyl sulfate [SDS], and 1:10 proteaseinhibitor cocktail [Sigma-Aldrich Corporate Offices, MO,Canada, http://www.sigmaaldrich.com]) and centrifuged at10,000 x g. The supernatant was recovered, and the proteinconcentration was determined using the Bradford method (7).Aliquots of 100 mg of total protein from the homogenizedthawed and unthawed tissue and thawed homogenate(kidney, n = 3; heart, n = 3; BAT, n= 3) were separated on a12% SDS polyacrylamide gel.Figure 1 - The A260/A280 ratios of the total RNA extracted fromthe thawed and unthawed tissue samples.Figure 2 - The 28S and 18S bands of the isolated total RNA from the control (unthawed) and thawed tissues. 1 and 2= control liver; 3and 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.Defrosted isolated total RNA is preservedOlivera IB et al.CLINICS 2012;67(3):255-259256The proteins were transferred onto a 0.20 mm pure nitrocel-lulose membrane (Bio-Rad Laboratories Inc, Hercules, CA,USA, http://www.bio-rad.com) for 1 h at 300 mA constantcurrent using a semidry transfer cell (Bio-Rad Laboratories Inc,Hercules, CA, USA, http://www.bio-rad.com). The gel wasstained with Coomassie blue to determine whether the proteinshad been transferred to the membrane. To ensure an adequateprotein transfer, the membranes were stained with Ponceau S.The blots were blocked overnight at 4 C˚ with 5% nonfat drymilk in Tris-buffered saline (1 mol/L Tris-HCl [pH 7.5] and3 mol/L NaCl).The membrane was washed with Tris-buffered saline plusTween (TBST) to remove the Ponceau S and then incubatedovernight at 4 C˚ with 1:5000 anti-beta-actin primary antibody(DakoDenmarkA/S, Glostrup, Denmark, http://www.dako.com).After washing the blots in TBST, theywere incubated with asecondary anti-mouse IgG-horseradish peroxidase-conjugatedantibody (Dako Denmark A/S, Glostrup, Denmark, http://www.dako.com) at 1:5000 for 2 hours. The blots were thenwashed again in TBST. The proteins were visualized using achemiluminescent agent (GE Healthcare, Piscatway, Uppsla,USA, http://www.gehealthcare.com) and autoradiographywith pre-flashed Kodak film (Eastman Kodak Company,Rochester, NY, USA, http://www.kodak.com).The intensity of the individual bands was quantified byoptical densitometry using Scion Image for Windows (ScionCorporation, Maryland, USA, http://scion-corporation.soft-ware.informer.com). A pre-stained protein marker (Bio-RadLaboratories Inc, Hercules, CA, USA, http://www.bio-rad.com) was used as the molecular weight standard.HistologyThe thawed tissues [pancreas (n=3), white (n=4) andbrown (n=3) adipose tissue, kidney (n=3), liver (n=3), andheart (n= 4)] were fixed in 10% buffered formalin andembedded in paraffin blocks. The morphological analysiswas performed as described (8). Briefly, 5-mm-thick sectionswere cut and stained with Masson’s trichrome and periodicacid Schiff for structural analyses. The tissue sections wereexamined using a magnification of 200X (Nikon InstrumentsInc, Melville, NY, USA, http://www.nikoninstruments.com).Statistical proceduresThe data are expressed as the mean¡ SEM. The normalityof the distributions was tested before performing Student’s tFigure 3A - The A260/A280 ratios of the total RNA in unthawed (control) and thawed isolated total RNA samples. Figure 3B - RNAconcentration in unthawed (control) and thawed isolated total RNA samples.Figure 4A - Integrity of the thawed and unthawed isolated total RNA samples. 1= ladder; 2 and 3=unthawed kidney (control); 4 to 18=thawed isolated total kidney RNA. Figure 4B - Renin mRNA level in thawed isolated total kidney RNA. Figure 4C - b-Actin mRNA level inthawed isolated total kidney RNA.CLINICS 2012;67(3):255-259 Defrosted isolated total RNA is preservedOlivera IB et al.257test with Welchs correction analysis. Values of p,0.05 wereconsidered statistically significant. The statistical analysiswas performed using GraphPad 4.00 for Windows (9).EthicsThe experiments in this study were previously approvedby the Ethics Committee of Hospital das Clinicas daFaculdade de Medicina da Universidade de Sa˜o Paulo.RESULTSA260/A280 ratio and the integrity of the total RNAfrom thawed tissuesThe A260/A280 ratio of the total RNA isolated from thethawed tissues was not affected relative to that of theunthawed tissues (Figure 1). However, when verifying theintegrity, the 28S and 18S bands were not observed,indicating that the total RNA was degraded (Figure 2).A260/A280 ratio, total RNA concentration, RNAintegrity and mRNA levels in thawed isolated totalRNA samplesAlthough the concentrations were the same (Figure 3B),the A260/A280 ratio was higher (p,0.05) for the thawedisolated total RNA than for the unthawed isolated totalRNA (Figure 3A). The integrity of the total RNA wasevaluated by gel electrophoresis analysis of the 28S and 18Sbands (Figure 4A), and this analysis confirmed the integrityof the isolated total RNA after defrosting. By RT-PCR, weobserved that the levels of b-actin (Figure 4B) and renin(Figure 4C) mRNA were perfectly maintained in the thawedisolated total RNA. From these data, it could be concludedthat thawed isolated total RNA can be analyzed andprocessed even after defrosting for up to 24 hours.Protein levelsThe b-actin protein levels in the tissue and homogenatewere analyzed by Western blotting, and these proteins werefound not to have been degraded due to the defrosting(Figure 5).HistologyWe found that the morphology of some of the thawedtissues was not preserved relative to that of the respectiveunthawed tissues (Figure 6).DISCUSSIONSome interesting and new observations were made in thepresent study. The samples used to determine the amount ofintact mRNA in previously isolated total RNA and the proteinlevels in thawed intact tissues and homogenates are wellpreserved even after more than twenty-four hours at roomtemperature. These results suggest that, in the event of atechnical problem with deep freezers that leads to defrosting,some material can still be used for some determinations.However, the histological structure and RNA extractedfrom the thawed tissues were not preserved.Okamoto et al. (10) have found that, during tissue freezingand thawing cycles, both the formation of ice crystals and theosmotic effect contribute to RNA degradation. In anotherstudy, it was found that RNA and DNA were stable only upto five hours after the tissue was excised when the tissue wasmaintained at room temperature (5). The results from thepresent study confirm that the RNA is not viable in defrostedtissues. The rapid degradation of RNA in tissues maintainedat room temperature or submitted to freezing and thawingcycles may be due to RNAase digestion. The preservation ofthe thawed previously isolated RNA found in the presentstudy supports this possible mechanism because RNAase isFigure 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).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).Defrosted isolated total RNA is preservedOlivera IB et al.CLINICS 2012;67(3):255-259258absent from these samples. Therefore, isolated total RNA isprotected from degradation due to thawing, maintaining theintegrity of expression analyses. Accordingly, it is recom-mended that total RNA be isolated as soon as possible aftertissue collection. The proteins in intact tissues are most likelydenatured due to prolonged storage at room temperature.However, the protein level was preserved in the thawedintact tissues and homogenates. A possible explanation forthis observation is that protein denaturation is inherent in theWestern blot technique.In conclusion, both gene and protein expression analysesmay be performed using total isolated RNA and intacttissue or homogenates, respectively, that have been storedfor many hours at room temperature.AUTHOR CONTRIBUTIONSOliveira IB performed the Western blotting and histology analysis andwrote the manuscript. Ramos DR, Lopes KL, and Souza RM performedthe PCR-RT analysis and helped to write the manuscript. Heimann JCrevised the manuscript. Furukawa LN conceived and coordinated the studyand wrote the manuscript.REFERENCES1. Kisand K, Kerna I, Kumm J, Jonsson H, Tamm A. Impact ofcryopreservation on serum concentration of matrix metalloproteinases(MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 inbiobank 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 oftransplanted 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 biomechanicalchanges in arteries: role of collagen matrix and smooth muscle cells. AnnBiomed 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, LiVolsiV, et al. Analysis of the molecular quality of human tissues. Am J ClinPathol. 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. Formalinfixation at low temperature better preserves nucleic acid integrity. PLoSONE. 2011;6:e21043, http://dx.doi.org/10.1371/journal.pone.0021043.7. Bradford MM. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. 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]. SanDiego, CA, USA: GraphPad Inc. http://www.graphpad.com.10. Okamoto M, Nakagata N, Toyoda Y. Cryopreservation and transport ofmouse spermatozoa at – 79 C˚. Exp Anim. 2001;50(1):83-86, http://dx.doi.org/10.1538/expanim.50.83.CLINICS 2012;67(3):255-259 Defrosted isolated total RNA is preservedOlivera IB et al.259

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

https://www.revistas.usp.br/clinics/article/download/19663/21727

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

Abstract

Similar works

Full text

Available Versions

Cadernos Espinosanos (E-Journal)