Further, we reported the levels of HBV DNA and HCV RNA in cancerous Nilotinib 641571-10-0 and noncancerous liver tissue using real-time detection (RTD)-PCR (34). RTD-PCR is an accurate assay method, but it can determine the levels of genomic DNA and RNA only in homogenized tissue. In this study, we developed a PCR-based in situ hybridization (PCR-ISH) method for detecting and visualizing HBV DNA, HBV RNA, and HCV RNA and comparing their protein expression patterns, with the aim to reveal the lobular distribution and intracellular localization of HBV and HCV in chronic liver disease and to clarify the state of persistent HBV and HCV infection in the liver. MATERIALS AND METHODS Patients. Twenty-nine patients were admitted to Tokyo Metropolitan Komagome Hospital for the treatment of hepatic tumors.
Of these patients, 14 were considered to have chronic HCV infection (persistently positive results for HCV antibody), 8 were diagnosed with chronic hepatitis B (persistently positive results for HBV surface antigen [HBsAg]), and 7 showed negative results for both viral markers but had metastatic liver cancer (6 with colonic cancer and 1 with gastric cancer). We used four samples from seven patients as controls for PCR-ISH and four samples from seven patients as controls for reverse transcriptase PCR (RT-PCR)-ISH (Table (Table1).1). Of the 14 patients with chronic hepatitis C, two showed positive results for HBV DNA by RTD-PCR. HBsAg and second-generation HCV antibody were measured by using enzyme-linked immunosorbent assay (ELISA) kits (Abbott Laboratories, Chicago, IL, and International Reagent Corp.
, Kobe, Japan, respectively). All 29 patients underwent hepatic resection. Histological evaluation of the liver was carried out according to the METAVIR scoring system (3). TABLE 1. Patient profiles and results of the present study Ethical approval. The Institutional Review Board of Tokyo Metropolitan Komagome Hospital approved the study. Written informed consent was obtained from all the subjects. Sample preparation. The liver tissue samples for HBV DNA detection were fixed in 10% buffered formalin (pH 7.4) for 18 h, embedded in paraffin, cut into 6-��m-thick sections, and mounted on silane-coated glass slides for use with a GeneAmp in situ PCR system 1000 unit (Applied Biosystems, Foster City, CA). The slides were washed thrice in xylene for 8 min at each washing, rinsed thrice in 99.
5% ethanol and 75% ethanol for 5 min at each rinsing, and rehydrated in distilled water for deparaffinization. For detecting HBV mRNA and HCV RNA, OCT-embedded frozen liver tissue samples were cut into 10-��m-thick sections and mounted on silane-coated glass slides. They were then fixed in 10% buffered formalin (pH 7.4) for 17 to 21 h, rinsed twice in distilled water treated with 0.01% diethylpyrocarbonate (DEPC) for 2 min GSK-3 at each rinse, rinsed in 99.5% ethanol for 1 min, and then air dried and stored at ?80��C until use.