Highly Sensitive Detection of HBV RNA in Liver Tissue by In Situ Hybridization
Highly Sensitive Detection of HBV RNA in Liver Tissue by In Situ Hybridization
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DOI：
10.1007/978-1-4939-6700-1_10
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Wieland, Stefan F.
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文献类型：
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作者：
Calabrese, Diego;Wieland, Stefan F.
一旦HBV被发现，在细胞水平上的HBV定位就成为研究HBV感染的工具。多年来，不仅在患者肝活检样本中，而且在许多其他实验系统中，已经开发出多种方法来检测细胞水平上的病毒抗原和核酸。最近，高灵敏度和特异性的原位杂交系统的发展使得在单拷贝水平上检测细胞mrna成为可能。采用这种系统（ViewRNA ISH组织检测；Affymetrix, Santa Clara， CA， USA）检测病毒RNA使我们能够可靠地鉴定丙型肝炎病毒感染患者肝脏中的丙型肝炎病毒RNA阳性细胞。同样，该方案能够在肝活检组织中检测到非常罕见的HBV阳性细胞。在这里，我们现在描述使用ViewRNA ISH系统检测HBV RNA的具体方案。该方案侧重于探针组设计，样品制备和数据解释，以准确检测肝组织样本中的HBV RNA。该方法简单明了，将在基础HBV病毒学研究和临床应用中非常有用。
As soon as HBV was identified, HBV localization at the cellular level became instrumental in studying HBV infection. Multiple methodologies for detection of viral antigens and nucleic acids at the cellular level, not only in patient derived liver biopsy samples, but also in many other experimental systems, have been developed over the years. Recently, the development of highly sensitive and specific in situ hybridization systems enabled detection of cellular mRNAs at the single copy level. Adaptation of such a system (ViewRNA ISH Tissue Assay; Affymetrix, Santa Clara, CA, USA) for the detection of viral RNAs allowed us to reliably identify hepatitis C virus RNA positive cells in the liver of HCV infected patients. Similarly, this protocol enabled detection of very rare HBV positive cells in liver biopsy tissue. Here, we now describe the specifics of the protocol for detection of HBV RNA using the ViewRNA ISH system. The protocol focuses on probe set design, sample preparation and data interpretation for accurate HBV RNA detection in liver tissue samples. The methodology is straightforward and will be very useful in the study of basic HBV virology as well as in clinical applications.