Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR
Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR
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DOI：
10.1038/labinvest.3700459
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发表时间：
2006-10-01
影响因子：
5
通讯作者：
Ballock, R. Tracy
中科院分区：
文献类型：
--
作者：
Shao, Yvonne Y.;Wang, Lai;Ballock, R. Tracy
生长板软骨细胞在体内的基因表达分析一直受到阻碍的固有困难，在矿化组织进行原位杂交。激光捕获显微切割和逆转录-聚合酶链反应（RT-PCR）的组合允许通过激光消融从组织切片中选择性去除细胞来分析基因表达。为了将该方法应用于矿化组织，需要脱钙过程。本研究的目的是确定激光捕获显微切割RT-PCR前组织脱钙的最佳方法，以保持mRNA群体的完整性。将丙酮、10%福尔马林和甲基卡林作为固定剂进行评价，而将Surgipath Decalicifier 1、10%乙二胺四乙酸（EDTA）和20% EDTA作为脱钙试剂进行评价。我们的研究结果表明，最佳的RNA质量被保存的脱钙协议由20%EDTA脱钙，然后在methacarn固定，虽然这种方法也与RNA量的减少。
The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier 1, 10% ethylenediaminetetraacetic acid ( EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.