title: Gene expression analysis in microdissected samples from decalcified tissues.
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.
  
  
  METHODS
  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.
authors.affiliation: Department of Prosthodontics and Periodontics, Division of Periodontics, School of Dentistry, Campinas State University, Piracicaba, São Paulo, Brazil. cris_salmon@yahoo.com.br
authors.forename: Cristiane Ribeiro
authors.initials: CR
authors.lastname: Salmon
authors.forename: Karina Gonzales
authors.initials: KG
authors.lastname: Silvério
authors.forename: Ana Paula de Oliveira
authors.initials: AP
authors.lastname: Giorgetti
authors.forename: Enilson Antonio
authors.initials: EA
authors.lastname: Sallum
authors.forename: Márcio Zaffalon
authors.initials: MZ
authors.lastname: Casati
authors.forename: Francisco Humberto
authors.initials: FH
authors.lastname: Nociti
mesh_terms: D000818:Animals; D018775:Bone Demineralization Technique; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D061346:Laser Capture Microdissection; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008334:Mandible / Q000737:chemistry / Q000378:metabolism; D008963:Molar / Q000737:chemistry / Q000378:metabolism; D010513:Periodontal Ligament / Q000737:chemistry / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
chemical_list: D002614:Chelating Agents; D012333:RNA, Messenger; D004492:Edetic Acid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases