Laser capture microdissection of rat periodontal ligament for gene analysis

Abstract

The periodontal ligament (PDL) is a connective tissue interposed between two hard tissues, viz., the root of a tooth and the alveolar bone, which makes it difficult to obtain directly. In the study reported here, PDL, subgingival connective tissue and pulp of rat molars were extracted directly by laser capture microdissection and the gene expression of TGF-β1 on the microdissected PDL was examined. The maxillae of rats were dissected and rapidly immersed in isopentane cooled with liquid nitrogen. Serial frontal sections of the rat first molar area were used for immunohistochemistry and for laser capture microdissection to localize the TGF-β1 gene. Gene expression and immunohistochemical localization of TGF-β1 also were examined in the pulp and subgingival connective tissues. TGF-β1 was located immunohistochemically in the fibroblasts in the PDL. A considerable amount of RNA was obtained by laser capture microdissection of these three tissues for analysis of gene expression. The reverse transcription- polymerase chain reaction demonstrated that glyceraldehyde 3-phosphate dehydrogenase was amplified in all three tissues, and that TGF-β1 was detected in the PDL. Laser capture microdissection makes it possible to analyze the gene expression of PDL and expression of TGF-β1in the PDL suggests that this gene could function in maintaining PDL.