In Situ hybridization and its diagnostic applications in pathology

Abstract

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.

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization

“…We did not observe unspecific binding, cross hybridization, or autofluorescence at a magnitude that disturbed the signal from Gallibacterium. It has been shown that these often hamper visualization of the hybridization signals in tissue (23). The oligonucleotides labeled with Cy3 and Cy5 used in the present study gave a clear bright signal, which enabled the use of narrow-band-pass filters, enhancing the signal-to-noise ratio considerably compared to those obtained with classical fluorophores, such as fluorescein and rhodamine derivatives, confirming the observations of Wessendorf and Brelje (36).…”

Amplification Methods to Increase the Sensitivity of In Situ Hybridization: Play CARD(S)

“…IN SITU HYBRIDIZATION (ISH) has proved to be an invaluable molecular tool in research and diagnosis and has significantly advanced the study of gene structure and expression at the level of individual cells in cell preparations as well as complex structural tissues (Dirks 1996;Forozan et al 1997;Hopman et al 1997;Lichter 1997;McNicol and Farquharson 1997). It has contributed substantially to the diagnosis and understanding of viral and neoplastic diseases and has provided invaluable insights into hormone regulation, storage, and secretion.…”

Histopathology for the Diagnosis of Infectious Diseases

“…In situ hybridization uses the complementary nature of nucleic acids, rather than an antibody, to impart specificity. [28,29] The nucleic acid probe, which may be labelled by a variety of methods, anneals to a specific target sequence in the microbial DNA or RNA.…”