Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues
Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues
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DOI：
10.1007/s004180050434
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发表时间：
2000-03-01
影响因子：
2.3
通讯作者：
Koji, T
中科院分区：
文献类型：
--
作者：
Shibata, K;Fujita, S;Koji, T
为了在钙化组织中原位分析特异性RNA表达的最佳性能，有必要选择合适的组织脱钙方案。我们评估了各种酸为基础的脱钙试剂的有用性，参考28 S rRNA染色使用胸腺嘧啶-胸腺嘧啶二聚体寡核苷酸探针原位杂交。评价的试剂是10%硝酸、10% HCl、5%甲酸、5%三氯乙酸、莫尔斯溶液、Plank-Rychlo溶液和K-CX溶液，所有这些都是常用的组织脱钙剂，并将它们对形态和RNA保留的影响与EDTA基溶液进行比较。以正常小鼠下颌骨为模型组织，用莫尔斯氏液、10%盐酸、Plank-Rychlo氏液和K-CX液脱钙后的切片中，成釉细胞形态保存较好，而用5%甲酸和莫尔斯氏液脱钙后的切片中，28 S rRNA的保留率最高。我们推荐使用莫尔斯溶液对待处理的组织进行脱钙，以快速分析特异性RNA表达。的确.在用莫尔斯氏液处理的切片中，我们检测到了强烈的特异性mRNA，定量分析表明28 S rRNA和特异性mRNA的信号强度比彼此相关，这取决于脱钙溶液。
For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse's solution, Plank-Rychlo's solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse's solution, 10% HCl, Plank-Rychlo's solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse's solution. We recommend Morse's solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed. we detected specific mRNAs strongly in sections treated with Morse's solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions.