Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays.
Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays.
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DOI：
10.1186/1477-3155-1-3
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发表时间：
2003-05-28
影响因子：
10.2
通讯作者：
Adam PJ
中科院分区：
文献类型：
--
作者：
Stamps AC;Terrett JA;Adam PJ
原发性疾病组织中疾病相关基因转录本的检测经常被非相关细胞类型的存在所混淆。直接在组织内检测基因表达的替代方法涉及抗体的产生，这可能是一个漫长的过程并且可能缺乏特异性，或者在组织切片中扩增逆转录的cDNA（原位RT-PCR）。后一种方法是高度特异性的，并且能够检测最初负责其合成的细胞中的转录物，但对组织结构具有高度破坏性，并且每次实验只能在一个或几个切片上进行，导致再现性低。在这项研究中，原位RT-PCR首次应用于市售的组织切片微阵列，使多达70个不同的样品同时检查。该技术的修改是详细的，保留可见的组织和细胞结构，并提高转录检测，同时防止显着产生的文物。
Detection of disease-associated gene transcripts in primary disease tissues is frequently confounded by the presence of non-involved cell types. Alternative methods of detecting gene expression directly within tissues involve either the generation of antibodies, which can be a lengthy process and may suffer from lack of specificity, or amplification of reverse-transcribed cDNA in tissue sections (in situ RT-PCR). The latter method is highly specific and enables detection of transcripts in the cells originally responsible for their synthesis, but is highly destructive of tissue structures and can be carried out on only one or a few sections per experiment, resulting in low reproducibility. In this study, in situ RT-PCR was applied for the first time to commercially available tissue section microarrays enabling the examination of up to 70 different samples simultaneously. Modifications to the technique are detailed that preserved visible tissue and cellular structures and improved transcript detection whilst preventing significant generation of artefacts.