Gene Expression Analysis in Microdissected Samples from Decalcified Tissues

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