Polymerase chain reaction in situ hybridization

Takahashi K, Wesselingh SL, Griffin DE, McArthur JC, Johnson RT, Glass JD (1996) Localization of HlV-1 in human brain using polymerase chain reaction/in situ hybridization and immuno-cytochemistry. Ann Neurol 39(6) 705-711... 

Nuovo, G. J., Becker, J., Simsir, A., Morgiotta, M., Khalife, G., and Shevchuk, M. (1994) HlV-1 nucleic acids localize to the spermatogonia and their progeny a study by polymerase chain reaction in situ hybridization. Am. J. Pathol. 144, 1142-1148. 

Wesselingh, S. L., Takahashi, K., Glass,J. D., McArthur,J. C., Griffin,J. W., and Griffin, D. E. (1997). Cellular localization of tumor necrosis factor mRNA in neurological tissue from HIV-infected patients by combined reverse transcriptase/polymerase chain reaction in situ hybridization and immunohistochemistry. J. Neuroimmunol. 74, 1—8. 

Matsuoka E, Takenouchi N, Hashimoto K, Kashio N, Moritoyo T, Higuchi I, Isashiki Y, Sato E, Osame M, Izumo S (1998) Perivascular T cells are infected with HTLV-I in the spinal cord lesions with HTLV-I-associated myelopathy/tropical spastic paraparesis Double staining of immunohistochemistry and polymerase chain reaction in situ hybridization. Acta Neuropathol (Berl) 96 340-346. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

ML Margall, N., Matias-Guiu, X., Chilton, M., Coll, P., Alejo, M., Nunes, V., Quilez, M., Rabella, N., Prats, G., and Prat, J., Detection of human papilloma virus 16 and 18 DNA in epithelial lesions of the lower genital tract by in situ hybridization and polymerase chain reaction. J. Clin. Microbiol. 31, 924-930 (1993). 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Ray, R., Komminoth, P., Machado, M., and Wolfe, H. J. (1991) Combined polymerase chain reaction and in situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Mod. Pathol. 4,124A. 

Drut, R. M Day, S., Drut, R., andMeisner, L. (1994) Demonstration of Epstein-Barr viral DNA in paraffin-embedded tissues of Burkitt s lymphoma from Argentina using the polymerase chain reaction and in situ hybridization. Fed. Pathol. 14,101-109. 

Finally, several PNA based methods for use in genetic diagnostics have been developed. Of particular interest are methods for fluorescence in situ hybridization (FISH), modulation of polymerase chain reaction (PCR) analyses, and detection of genetic material by sensors and mass spectrometry. 8 ... 

The first binding studies on solubilized membranes from rat brain demonstrated the presence of a low affinity adenosine receptor with characteristics of the A3 subtype (Oliveira et al. 1991). However, in situ hybridization studies in the rat indicated the presence of A3AR mRNA only in the testis (Meyerhof et al. 1991a Zhou et al. 1992 Rivkees 1994) and not in the CNS (Rivkees et al. 2000). Similarly, no expression of A3AR in the brain of mice or in the hippocampi of humans was detected (Rivkees et al. 2000). However, by reverse transcription-polymerase chain reaction (RT-PCR)... 

However, be aware that in spite of the usefulness of the MIB-1 antibody in assessing the rate of cell proliferation, the classification of cancers (e.g., breast cancer) by the size of the primary tumor and the presence and extent of lymph node metastases does not adequately explain differences in the clinical outcome of individual patients. Cell proliferation indices are commonly used, along with other diagnostic parameters, to estimate the risk of recurrence of a cancer for individual patients. Therefore, it is important to understand the relationship between various indices of proliferation such as MIB-1 labeling index and detection by either in situ hybridization or polymerase chain reaction. This approach will lead to quality assurance in diagnosis. 

A reliable method of measuring the ER content in human breast cancer is important for optimal treatment and a qualified estimate of the recurrence-free survival of the patient. The majority of the studies on the expression of ERs, especially ER 3 in human tissues, have been accomplished using RNA techniques such as reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Although the RT-PCR method is an effective tool to describe the presence of a particular gene in the tissue, this approach does not indicate the specific cell that expresses the gene. 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Naked plasmid DNA was not only trapped in the cytoplasm around the site of microinjection, as visualized by fluorescence in situ hybridization (FISH) or using FITC-labeled DNA, but was also eliminated rapidly at physiological temperature (Lechardeur et al., 1999). The disposal of the DNA was completely prevented when the cells were kept at 4°C (Lechardeur et al., 1999). A similar conclusion was reached by monitoring the amount of microinjected expression cassette by the polymerase chain reaction (PCR) (Pollard et al., 2001), suggesting that the metabolic instability of naked DNA contributes to the low efficiency of gene transfer (Lechardeur et al., 1999 Mirzayans et al, 1992 Neves etal., 1999 Pollard et al., 2001). 

W3. Weiss, L. M., Chen, Y. Y., Liu, X. F., and Shibata, D., Epstein-Barr virus and Hodgkin s disease. A correlative in situ hybridization and polymerase chain reaction study. Am. J. Pathol. 139, 1259-1265 (1991). 

The 5-HT6 receptors are positively coupled to adenylate cyclase. In the absence of selective agonists and antagonists as well as radioligands, they have been initially localized in the rat brain by Northern blot analyses (218,219), in situ hybridization histochemistry (219-221), and quantitative reverse transcription followed by polymerase chain reaction (222) (see also ref. 210). These studies have established that the receptor mRNA is abundant in extrapyramidal areas such as the striatum, as well as in limbic areas such as the nucleus accumbens, olfactory tubercle, hippocampus, and hypothalamus. 

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