Gene Expression Analysis in Microdissected Samples from Decalcified Tissues

dc.creatorSalmon, CR
dc.creatorSilverio, KG
dc.creatorGiorgetti, APD
dc.creatorSallum, EA
dc.creatorCasati, MZ
dc.creatorNociti, FH
dc.date2012
dc.dateJUN
dc.date2014-07-30T17:47:33Z
dc.date2015-11-26T16:52:03Z
dc.date2014-07-30T17:47:33Z
dc.date2015-11-26T16:52:03Z
dc.date.accessioned2018-03-28T23:38:56Z
dc.date.available2018-03-28T23:38:56Z
dc.identifierDiagnostic Molecular Pathology. Lippincott Williams & Wilkins, v. 21, n. 2, n. 120, n. 126, 2012.
dc.identifier1052-9551
dc.identifierWOS:000304112400008
dc.identifier10.1097/PDM.0b013e31823e9395
dc.identifierhttp://www.repositorio.unicamp.br/jspui/handle/REPOSIP/67835
dc.identifierhttp://repositorio.unicamp.br/jspui/handle/REPOSIP/67835
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1276167
dc.descriptionObjective: 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.
dc.description21
dc.description2
dc.description120
dc.description126
dc.languageen
dc.publisherLippincott Williams & Wilkins
dc.publisherPhiladelphia
dc.publisherEUA
dc.relationDiagnostic Molecular Pathology
dc.relationDiagn. Mol. Pathol.
dc.rightsfechado
dc.sourceWeb of Science
dc.subjectperiodontal ligament
dc.subjectlaser capture microdissection
dc.subjectgene expression
dc.subjectLaser-capture Microdissection
dc.subjectParaffin-embedded Tissue
dc.subjectRna Extraction
dc.subjectRt-pcr
dc.subjectOptimization
dc.subjectCells
dc.subjectBone
dc.titleGene Expression Analysis in Microdissected Samples from Decalcified Tissues
dc.typeArtículos de revistas


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