Biotinylated probes for in situ hybridization histochemistry: use for mRNA detection.

Bloch, Bertrand

doi:10.1177/41.12.8245422

Cited by 24 publications

(7 citation statements)

References 21 publications

(37 reference statements)

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“…Non-isotopic labelling-The first non-isotopic label was biotin, 68,69 and this continues to be used because of the high sensitivity of streptavidin detection systems. However, the widespread presence of endogenous biotin and the limited success of blocking methods 70 have stimulated the development of a range of other labels.…”

Section: Probe Labelling and Detectionmentioning

confidence: 99%

In Situ hybridization and its diagnostic applications in pathology

1997

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In situ hybridization (ISH) is a technique by which specific nucleotide sequences are identified in cells or tissue sections. These may be endogenous, bacterial or viral, DNA or RNA. On the basis of research applications, the technique is now being translated into diagnostic practice, mainly in the areas of gene expression, infection and interphase cytogenetics. Diagnostic applications are most often based on short nucleotide sequences (oligomers) labelled with non‐isotopic reporter molecules, and sites of binding may be localized by histochemical or immunohistochemical methods. The technique can be applied to routinely fixed and processed tissues; with some targets, it is even possible to obtain hybridization in autopsy material. ISH has been used to detect messenger RNA (mRNA) as a marker of gene expression, where levels of protein storage are low; for example, to confirm an endocrine tumour as the source of excess hormone production. Its application in infectious diseases has to date been mainly in viral infections, such as the typing of human papillomavirus (HPV) or the detection of Epstein–Barr virus by the presence of small nuclear RNAs (EBERs). The expression of mRNAs for histone proteins has been used to detect cells in S phase, and related methods may be applied to detect apoptotic cells. Using probes to chromosome‐specific sequences, it is possible to detect aneuploidy, and to document changes in specific chromosomes, which may have prognostic significance in some tumours, such as B‐cell chronic lymphatic leukaemia. Using sequence‐specific probes, translocations can be identified, such as the t(11;12) of Ewing's sarcoma. This review presents an outline of the technique of in situ hybridization and discusses areas of current and potential diagnostic application. © 1997 John Wiley & Sons, Ltd.

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Section: Probe Labelling and Detectionmentioning

confidence: 99%

In Situ hybridization and its diagnostic applications in pathology

1997

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“…In situ hybridization was performed according to Bloch (1993). A linearized plasmid containing the region 4041-4840 of HCV flanked by promoter sequences for T7 and T3 RNA polymerases was used to transcribe digoxigenin-labelled sense and antisense riboprobes using Riboprobe Combination System T3/T7 (Promega), according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice

Alonzi

Agrati

Costabile

et al. 2004

Journal of General Virology

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To assess the effects of constitutive hepatitis C virus (HCV) gene expression on liver, transgenic mice carrying the entire HCV open reading frame inserted in the a1 antitrypsin (A1AT) gene were generated. Expression of A1AT/HCV mRNA was found to be mainly limited to perivascular areas of the liver as indicated by in situ hybridization analysis. HCV core protein was detected in Western blots of liver extracts, whereas the expression of E2, NS3 and NS5 proteins was revealed by immunostaining of liver samples using HCV-specific antisera. Histological analysis of HCV transgenic mice showed that these animals develop extensive steatosis, but very little necrosis of liver tissue. Moreover, a consistent T cell infiltrate and a slight hepatocyte proliferation were observed. Phenotypic analysis of cells infiltrating the liver indicated that recruitment and/or expansion of residing CD8 + , NK, NKT and cd T cells occurred in transgenic animals. Among these cells, a large fraction of CD8 + T lymphocytes released mainly IL-10 and, to a lesser extent, IFN-c upon mitogenic stimulation in vitro. Furthermore, both intrahepatic lymphocytes and splenocytes did not produce cytokines in response to HCV antigens. Thus, these data indicate that constitutive expression of HCV proteins may be responsible for intrahepatic lymphocyte recruitment in absence of viral antigen recognition. This response is likely to be driven by virus-induced cellular factors and may play a significant role in the immunopathology of chronic HCV infection and liver disease. INTRODUCTIONHepatitis C virus (HCV) is a member of the Flaviviridae and is the major cause of non-A, non-B hepatitis (Choo et al., 1989;Houghton et al., 1991;Kuo et al., 1989). HCV has a single-stranded RNA genome of positive polarity of about 9?5 kb that contains a long open reading frame (ORF) encoding a polyprotein of approximately 3000 amino acids Kato et al., 1990;Takamizawa et al., 1991). The polyprotein precursor has the following gene order: NH 2 C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b COOH (Eckart et al., 1993;Grakoui et al., 1993; Hijikata et al., 1993a, b;Lin et al., 1994;Tomei et al., 1993) C is an RNA-binding protein (Santolini et al., 1994) and is considered to be the viral nucleocapsid; E1 and E2 are thought to be the virion glycoproteins; p7 is a protein involved in the formation of ion channels (Griffin et al., 2004) and is inefficiently cleaved from the E2 polypeptide (Mizushima et al., 1994). Processing of the structural polypeptides is mediated by cellular signal peptidase (Hijikata et al., 1993b;Santolini et al., 1994). The HCVencoded metalloproteinase and serine protease located in the NS2 to NS3 region and N-terminal one-third of NS3 protein, respectively, mediate cleavages in the nonstructural region (Clarke, 1997).More than 170 million people worldwide are infected with HCV, and the complications of liver cirrhosis and hepatocellular carcinoma cause significant morbidity and mortality. The virus is cleared in a minority of patients and 70-80 % develop chronic infec...

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“…Results obtained with biotin-based systems should therefore be interpreted with care. Although the biotin system has been used extensively (Bloch 1993;Mitchellet al 1993;Le Guellec et al 1991;Guitteny et al 1989), few authors have discussed interference by endogenous biotin (Varma et al 1994;Yao et al 1993). The detection of mitochondrial rRNA with a biotin-based system (Escaig-Haye et al 1991;Binder et al 1986) therefore requires additional analysis by studies of endogenous biotin which is specially concentrated in mitochondriae, associated with the Krebs cycle enzymes.…”

Section: Pros and Cons Of Biotin And Digoxigenin In The Detection Systemmentioning

confidence: 99%

Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand?

Chevalier

Michel

et al. 1997

J Histochem Cytochem.

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Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level.

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Biotinylated probes for in situ hybridization histochemistry: use for mRNA detection.

Cited by 24 publications

References 21 publications

In Situ hybridization and its diagnostic applications in pathology

In Situ hybridization and its diagnostic applications in pathology

Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice

Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand?

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