RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation

Abstract

The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.