Detection of human papillomavirus in cervical scrapings by in situ hybridization and the polymerase chain reaction in relation to cytology

Summary A gynaecological out-patient population consisting of 200 patients aged 19–43 years (mean age 34.2 years) was screened for the presence of human papillomavirus (HPV) by the polymerase chain reaction and in situ hybridization on cervical scrapings. A novel method was applied for the detection of HPV in cervical cells by embedding them in a paraffin block before in situ hybridization was performed. This technique resulted in well preserved cytological morphology, easy performance and economy of probes. In eight of the 200 patients (4%), human papillomavirus DNA was revealed by the polymerase chain reaction. Subtyping revealed the presence of HPV serotype 16 DNA in three of these patients. In one patient HPV serotype 18 DNA was also present. The in situ hybridization assay was able to detect all those cases with a specific HPV serotype infection.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Polymerase chain reaction in situ: an appraisal of an emerging technique

Summary Polymerase chain reaction (PCR)in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCRin situ is, however, technically difficult, and amplification of the target DNA is only 30–300 fold. In this article we present an overview of PCRin situ techniques used to amplify both DNA and RNA targets (RT-PCRin situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks byTaq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.     

Fluorescein as a label for non-radioactive in situ hybridization

Summary Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.     

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.     

Discrimination of closely homologous HPV types by nonisotopicin situ hybridization: definition and derivation of tissue melting temperatures

Summary It is generally assumed that nucleic acid association duringin situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 byin situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies.By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tm^t) were derived. The corresponding values for Tm^t are 10–20°C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing.     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

A practical approach to FRET-based PNA fluorescence in situ hybridization

Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location, and dynamics but also potentially a wide variety of close range RNA–RNA interactions. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results.     

Localization of mRNA for testis-specific histone H1t by in situ hybridization

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.     

Estimating the initial reaction velocity of a soluble dehydrogenase in situ

Summary The initial reaction velocities (v _i ) of lactate dehydrogenase in single hepatocytes were determined, by microdensitometry or computer-assisted image analysis, in sections of unfixed mouse liver incubated at 37°C on substrate-containing agarose gel films. They were found to fit the equations v _i =2.82°A and v _i =v+2°A, where v and °A are, respectively, gradients (or steady-state linear velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) on incubation time plots for incubation times between 1 and 3 min. Both equations were independent of section thickness between 4 and 14 m. The observed and calculated values of v _i , agreed within 11.5% (n = 71). The validity of the equations for v _i was confirmed by showing that the calculated v _i was proportional to the thickness of the section and hence the amount of enzyme present. Thus, v _i can be determined from measurements of either °A alone or v and °A.     

CCD microscopy and image analysis of cells and chromosomes stained by fluorescence in situ hybridization

Summary This paper reviews methods and applications of CCD microscopy for analysing cells and chromosomes subjected to fluorescence in situ hybridization (FISH). The current status of indirect and direct FISH staining methods with respect to probe labelling, detection sensitivity, multiplicity and DNA resolution is summarized. Microscope hardware, including special multi-band pass filters and CCD cameras required for FISH analysis, is described. Then follows a detailed discussion of current and emerging applications such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells), comparative genomic hybridization, automated evaluation of radiation-induced chromosomal translocations, and high-resolution DNA mapping on highly extended chromatin. Finally, the limitations of the present methodology and future prospects are discussed.The Historical Journal lecture for 1993 presented by Dr Tanke to a joint meeting of the Royal Microscopical society of cell Biology, Clinical Cytology, Electron Microscopy and Pathology and the Belgian Socities in Brussels, Belgium on 2 September 1993.This work was supported financially by the Netherlands Organization for Scientific Research (NWO) and in part by Imagenetics, Framingham, MA, USA.     

原位PCR技术及其应用前景

原位PCR是分子生物学领域中一种崭新的技术,它结合了PCR技术和原位杂交技术的优点.该文介绍了原位PCR技术的起源、发展及方法学,并简要描述了该技术的应用现状及前景．     

The detection of the mRNAs of procollagen types I, II and III in human fetal fingers by in situ hybridization using digoxigenin-labelled oligonucleotide probes

Summary Messenger RNAs (mRNAs) encoding procollagen 1 type I, 1 type II and 1 type III have been localized in paraffin sections of human fetal fingers using digoxigenin-labelled synthetic oligonucleotide probes. The probe-mRNA hybrids were visualized using an anti-digoxin antibody amplified with sandwich techniques. These protocols provided an excellent hybridization signal with minimal background noise. The sensitivity of the protocols was nearly equivalent to that seen when using isotopic cDNA probes. In human fetal fingers, intense hybridization signals for procollagen 1 type I mRNA were detected in the osteoblasts and the fibroblasts of periosteum and perichondrium, the tenocytes of tendons, fibroblasts of ligaments, the synovial membrane and deeper layers of the dermis. In contrast, positive hybridization signals for procollagen 1 type II mRNA were visualized in chondrocytes and the cambial layer of perichondrium. The signals for procollagen 1 type III mRNA were detected in the fibroblasts of the dermis and perichondrium. The probes which have lower melting temperatures (Tm) could not detect the corresponding mRNAs.