Localization of hepatitis C virus RNA in human liver biopsies by in situ hybridization using thymine-thymine dimerized oligo DNA probes: improved method.
To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.
Endo
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First Department of Internal Medicine, Okayama University Medical School, Japan.
Yamada
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Nakane
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Tsuji
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T
D001483:Base Sequence; D001706:Biopsy; D003851:Deoxyribonucleases / Q000494:pharmacology; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008099:Liver / Q000382:microbiology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011740:Pyrimidine Dimers; D012367:RNA, Viral / Q000032:analysis*
D015345:Oligonucleotide Probes; D011740:Pyrimidine Dimers; D012367:RNA, Viral; D003851:Deoxyribonucleases