The In Situ Detection of PCR-Amplified Hepatitis C RNA

Abstract

The discovery of PCR in 1985 has had an enormous impact on the field of molecular diagnostics. However, an important drawback of solution-phase PCR is that one cannot localize the target of interest to a specific cell type owing to the obligatory tissue destruction required for DNA extraction. This problem has been circumvented by the field of in situ PCR. This chapter will describe the theoretical basis of in situ PCR using hepatitis C as a model system. The goals of this manuscript are:

• 1. To describe the various DNA synthesis pathways that may be operative during in situ PCR.

• 2. To describe the key preparatory steps of reverse transcriptase (RT) in situ PCR

• 3. To describe the actual protocol of RT in situ PCR

• 4. To discuss potential problems when doing RT in situ PCR and how to resolve these problems, using hepatitis C infection of liver tissues as the model system