Detection of specific mRNAs in routinely processed dermatopathology specimens.
Detection of specific mRNAs in routinely processed dermatopathology specimens.
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检测常规处理的皮肤病理学标本中的特定 mRNA。
DOI：
10.1097/00000372-199510000-00008
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Longley,J
中科院分区：
文献类型：
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作者：
Tyrrell,L;Elias,J;Longley,J
为了确定不同固定剂对组织病理学存档标本中mRNA的回收和检测的影响，将正常皮肤活检标本固定在中性和酒精缓冲的福尔马林、丙酮、Carney固定剂、methacarn和布因溶液中。常规处理组织，并将切片封固用于原位杂交或脱蜡用于RNA提取。使用随机六聚体逆转录提取的mRNA，并使用甘油醛-3-磷酸脱氢酶（GAPDH）和β-肌动蛋白特异性引物通过聚合酶链式反应扩增所得cDNA。除布因固定液外，所有固定液处理的组织均可通过凝胶电泳检测到GAPDH和肌动蛋白的扩增产物。自1990年以来，福尔马林固定，石蜡包埋存档活检的平行研究也得到了类似的结果。丰度较低的mRNA，如编码白细胞介素-11或T细胞受体p链的mRNA，可通过Southern印迹和标记寡核苷酸探针杂交或通过克隆和测序检测。使用寡核苷酸探针的原位杂交研究在福尔马林固定的组织中最为成功，包括实验固定的组织和存档活检样本。因此，mRNA可以从福尔马林固定、石蜡包埋的档案材料中分离并定位。由于皮肤病理学实验室档案通常包含来自广泛疾病的样本，这些样本可以在未经人类调查委员会批准的情况下访问，因此这些实验室代表了研究皮肤中基因调控和表达的逻辑起点。
To determine the effect of different fixatives on the recovery and detection of mRNAs from archival histopa-thology specimens, biopsies of normal skin were fixed in neutral and alcohol-buffered formalin, acetone, Carney's fixative, methacarn, and Bouin's solution. Tissue was routinely processed, and sections were either mounted for in situ hybridization or deparaffinized for RNA extraction. Extracted mRNA was reverse-transcribed using random hexamers, and the resulting cDNA was amplified by the polymerase chain reaction using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and [beta]-actin. Amplification products of both GAPDH and actin could be detected by gel electrophoresis from tissues processed in all fixatives except Bouin's. A parallel study of formalin-fixed, paraffin-embedded archival biopsies accessioned since 1990 gave similar results. Less abundant mRNAs, such as those encoding interleukin-11 or the T-cell receptor p-chain, could be detected by Southern blotting and hybridization with labeled oligonu-cleotide probes or by cloning and sequencing. In situ hybridization studies using oligonucleotide probes were most successful with tissue fixed in formalin, including both the experimentally fixed tissues and the archival biopsy samples. Thus, mRNAs may be isolated from and localized in formalin-fixed, paraffin-embedded archival material. Because dermatopathology laboratory archives typically contain samples from a wide spectrum of diseases that can be accessed without Human Investigation Committee approval, these laboratories represent a logical starting point for studying gene regulation and expression in skin.