ABSTRACT
OBJECTIVE: The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples. DESIGN: Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase. RESULTS: Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified. CONCLUSIONS: Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.
Subject(s)
Gene Expression Profiling , Laser Capture Microdissection , Animals , Bone Demineralization Technique , Chelating Agents/chemistry , Edetic Acid/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Liver/chemistry , Liver/metabolism , Mandible/chemistry , Mandible/metabolism , Molar/chemistry , Molar/metabolism , Periodontal Ligament/chemistry , Periodontal Ligament/metabolism , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Tissue FixationABSTRACT
Objetivo: Avaliar a resistência da união de sistemas adesivos à dentina contaminada por cimentos temporários com ou sem eugenol. Método: Foram obtidas superfícies planas de dentina de 24 terceiros molares humanos. Com exceção do grupo controle (n=8), as superfícies foram cobertas com Interim Restorative Material (Caulk Dentsplay, Milford, DE, USA) ou Cavit (3M ESPE, St. Paul, MN, EUA) e mantidas em estufa a 37°C por sete dias. Após a remoção dos cimentos, os sistemas adesivos Adper Single Bond (3M ESPE, St. Paul, MN, EUA) ou Clearfil SE Bond (Kuraray Co. Ltd., Osaka, Japão) foram aplicados segundo a recomendação dos fabricantes e, em seguida, realizadas as construções de coroas em resina composta. Os dentes foram secionados em espécimes com área transversal de união de 0,81mm², os quais foram submetidos ao teste de microtração em máquina para ensaios mecânicos com velocidade do atuador de 0,5mm/min. Os dados foram analisados por testes t e Anova, complementada por testes de Tukey (α=0,05). Resultados: Para Adper Single Bond (3M ESPE, St. Paul, MN, EUA), a resistência de união foi estatisticamente não-diferente (p>0,05) para todas as condições experimentais. Para Clearfil SE Bond (Kuraray Co. Ltd., Osaka, Japão), apenas o grupo Interim Restorative Material (Caulk Dentsplay, Milford, DE, USA) apresentou resistência de união significativamente inferior (30,1±13,8 MPa) em relação aos demais grupos; controle (38,9±13,5 MPa) e Cavit (3M ESPE, St. Paul, MN, EUA) (42,1±11,0 MPa), os quais não apresentaram diferença significativa entre si. Conclusão: Concluiu-se que o recobrimento prévio da dentina com cimento temporário, contendo eugenol, exerceu efeito deletério apenas no desempenho adesivo do sistema autocondicionante.
Objective: The aim of this study was to assess the bond strength of adhesive systems to dentin contaminated by temporary cements with or without eugenol. Method: Flat dentin surfaces were obtained from twenty-four human third molars. With exception of the control group (n=8), the surfaces were covered with Interim Restorative Material (Caulk Dentsplay, Milford, DE, USA) or Cavit (3M ESPE, St. Paul, MN, USA) and kept in an oven at 37°C for seven days. After removing the cements, the adhesive systems Adper Single Bond (3M ESPE, St. Paul, MN, USA) or Clearfil SE Bond (Kuraray Co. Ltd., Osaka, Japan) were applied in accordance with the manufacturers' recommendations, and then the crowns were constructed in of resin composite. The teeth were sectioned into specimens with a cross-sectional bond area of 0.81mm², which were submitted to microtensile testing in a mechanical test machine at an actuator speed of 0.5mm/min. The data were analyzed by t- and ANOVA tests, complemented by Tukey tests (α=0.05). Results: For Adper Single Bond (3M ESPE, St. Paul, MN, USA), bond strength did not differ statistically (p>0.05) for all the experimental conditions. For Clearfil SE Bond (Kuraray Co. Ltd., Osaka, Japan), only the Interim Restorative Material (Caulk Dentsplay, Milford, DE, USA) Group showed significantly lower bond strength (30.1±13.8 MPa) in comparison with the other groups; control (38.9±13.5 MPa) and Cavit (3M ESPE, St. Paul, MN, USA) (42.1±11.0 MPa), which showed no significant difference between them. Conclusion: It was concluded that the previous covering of dentin with temporary cement containing eugenol had a deleterious effect on the adhesive performance of the self-etching system only.
