RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation
RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation
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DOI：
10.1016/j.archoralbio.2005.06.010
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发表时间：
2006-02-01
影响因子：
3
通讯作者：
Putnins, EE
中科院分区：
文献类型：
--
作者：
Ekuni, D;Firth, JD;Putnins, EE
目的：口腔软组织的结构组织及其与高度钙化牙齿的关系很难保存，除非组织脱钙，石蜡包埋，然后切片。然而，釉质脱钙时间及其对RNA完整性的负面影响使得有效地分析原位基因表达变得困难。本研究探讨了微波强化脱钙对处理时间、RNA完整性以及软硬组织中角质形成细胞生长因子受体、硬化轴和骨连蛋白细胞类型特异性标志物原位mRNA表达的影响。设计：取Wistar大鼠颌骨和下颌骨。右侧组织用微波+10%EDTA溶液(M+)脱钙，左侧组织用10%EDTA溶液(M-)单独脱钙。结果：与M组相比，微波组脱钙时间缩短50%，对组织形态、RNA质量和基因表达的原位检测无显著影响。结论：微波脱钙石蜡包埋口腔组织原位RT-PCR基因表达是一种在保持良好软硬组织结构的同时进行基因原位表达定位的有效技术。(C)2005爱思唯尔有限公司。保留所有权利。
Objective: 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.Design: Maxillas and mandibles were obtained from three mate 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-).Results: 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.Conclusions: 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. (c) 2005 Elsevier Ltd. All rights reserved.