LAUSR

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.