In situ hybridisation (ISH) is based on the complementary pairing of labelled DNA or RNA probes with normal or abnormal nucleic acid sequences in intact chromosomes, cells or tissue sections. Compared with other molecular biology techniques applicable to anatomical pathology, ISH enjoys better rapport with histopathologists because of its similarity to immunohistochemistry. It has the unique advantage over other molecular biology techniques--largely based on probe hybridisation with nucleic acid extracted from homogenised tissue samples--of allowing localisation and visualisation of target nucleic acid sequences within morphologically identifiable cells or cellular structures. Probes for ISH may bear radioactive or non-radioactive labels. Isotopic probes (3H, 32P, 35S, 125I) are generally more sensitive than non-isotopic ones but are less stable, require longer processing times and stringent disposal methods. Numerous non-isotopic labels have been used; of these biotin and digoxigenin are the reporters of choice. Optimised non-isotopic systems of equivalent sensitivity to those which use radioactive-labelled probes have been described. In ISH, finding the optimal balance between good morphological preservation of cells and strong hybridisation signals is crucial. Tissue fixation and retention of cytoskeletal structures, unfortunately, impede diffusion of probes into tissues. ISH sensitivity is also influenced by inherent properties of the probe and hybridisation conditions. Although ISH is largely a research tool, it is already making strong inroads into diagnostic histopathology. It has been applied for the detection of various infective agents particularly CMV, HPV, HIV, JC virus, B19 parvovirus, HSV-1, EBV, HBV, hepatitis delta virus, Chlamydia trachomatis, salmonella and mycoplasma in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)
Matched MeSH terms: In Situ Hybridization/methods*
Nine simple sequence repeat (SSR) markers were developed from Shorea curtisii using two different methods. One SSR locus was isolated by the commonly used method of screening by colony hybridization, and the other eight loci were isolated by a vectorette PCR method. Primer pairs were designed based on the sequences of all these SSR loci. Analysis of 40 individuals of S. curtisii from natural forest in Malaysia revealed that all SSR loci were polymorphic. Four SSR markers, Shc01, Shc04, Shc07 and Shc09, were highly polymorphic. We have also tested the applicability of these SSR printers to other species of Dipterocarpaceae using PCR amplification. Because the flanking region sequences of the S. curtisii SSRs were well conserved within this family, the SSR primers for S. curtisii can be applied to almost all species of Dipterocarpaceae.
In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.
Matched MeSH terms: In Situ Hybridization/methods*
In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.
Matched MeSH terms: In Situ Hybridization/methods*
Circular RNAs characterize a class of widespread and diverse endogenous RNAs which are non-coding RNAs that are made by back-splicing events and have covalently closed loops with no polyadenylated tails. Various indications specify that circular RNAs (circRNAs) are plentiful in the human transcriptome. However, their participation in biological processes remains mostly undescribed. To date thousands of circRNAs have been revealed in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies specify that these transcripts control expression of protein-coding linear transcripts and thus encompass a key component of gene expression regulation. This chapter provide a comprehensive overview on functional validation of circRNAs. Furthermore, we discuss the recent modern methodologies for the functional validation of circRNAs such as RNA interference (RNAi) gene silencing assay, luciferase reporter assays, circRNA gain-of-function investigation via overexpression of circular transcript assay, RT-q-PCR quantification, and other latest applicable assays. The methods described in this chapter are demonstrated on the cellular model.