In situ reverse transcription‐polymerase chain reaction. Applications for light and electron microscopy
Identifieur interne : 003B56 ( Main/Exploration ); précédent : 003B55; suivant : 003B57In situ reverse transcription‐polymerase chain reaction. Applications for light and electron microscopy
Auteurs : Gérard Morel [France] ; Martina Berger [France] ; Brice Ronsin [France] ; Sophie Recher [France] ; Sylvie Ricard-Blum [France] ; Hichem C. Mertani [Singapour] ; Peter E. Lobie [Singapour]Source :
- Biology of the Cell [ 0248-4900 ] ; 1998-03.
English descriptors
- Teeft :
- Alkaline phosphatase, Amplification, Amplification mixture, Amplification products, Amplification reaction, Annealing, Appropriate controls, Avian myeloblastosis virus, Bagasra, Biological material, Boca york, Boehringer mannheim, Careful optimization, Cdna, Cell membrane, Cell suspensions, Cycle number, Denaturation, Detection system, Diffusion artifacts, Digestion, Digestion step, Direct reaction, Dnase, Dnase digestion, Dntps, Electron microscopy, Elongation step, Embedding, Endocrine cell populations, Endogenous biotin, Ethanol, Extension time, Final concentration, First step, Fixation, Fixation time, Fixative, Gene expression, Gene rearrangements, Genome, Glass slides, Gold particles, Good preservation, Growth hormone receptor mrna, Haase, High number, Higher concentration, Host cell, Human papillomavirus, Human press, Humid chamber, Hybridization, Hybridization step, Hybridization temperature, Hydrophylic resin, Indirect reactions, Individual cells, Infectious agents, Komminoth, Light microscopy, Localization, Major pitfall, Mertani, Mispriming, Molecular biology, Molecular diagnosis, Morel, Mrna, Mrna coding, Negative strand, Nucleic, Nucleic acid, Nucleic acid sequences, Nucleic acid target, Nucleic acids, Nucleotide, Nuovo, Paraformaldehyde, Phosphate buffer, Pituitary gland section, Polymerase, Polymerase activity, Polymerase chain reaction, Positive cells, Positive strand, Possible adsorption, Possible degradation, Powerful technique, Primer, Proteolytic digestion, Protocol, Raven press, Receptor, Replication, Rnase, Ronsin, Room temperature, Second step, Secondary structures, Several approaches, Signal density, Target sequence, Tissue preparation, Tissue section, Tissue sections, Total volume, Transcriptase, Ultrathin, Ultrathin sections, Unlabeled nucleotides, Unlabeled probe, Vibratome sections, Viral, Viral genome, Viral methods, Visna virus, White resin.
Abstract
Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell‐localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT‐PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl2, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT‐PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.
Url:
- https://api.istex.fr/ark:/67375/WNG-XT87NFNB-2/fulltext.pdf
- https://api.istex.fr/ark:/67375/6H6-G281DK2K-F/fulltext.pdf
DOI: 10.1016/S0248-4900(98)80335-3
Affiliations:
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Mertani</name><affiliation wicri:level="4"><country xml:lang="fr">Singapour</country><wicri:regionArea>Institute of Molecular and Cell Biology, National University of Singapore</wicri:regionArea><orgName type="university">Université nationale de Singapour</orgName></affiliation></author><author><name sortKey="Lobie, Peter E" sort="Lobie, Peter E" uniqKey="Lobie P" first="Peter E." last="Lobie">Peter E. Lobie</name><affiliation wicri:level="4"><country xml:lang="fr">Singapour</country><wicri:regionArea>Institute of Molecular and Cell Biology, National University of Singapore</wicri:regionArea><orgName type="university">Université nationale de Singapour</orgName></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biology of the Cell</title><title level="j" type="alt">BIOLOGY OF THE CELL</title><idno type="ISSN">0248-4900</idno><idno type="eISSN">1768-322X</idno><imprint><biblScope unit="vol">90</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="137">137</biblScope><biblScope unit="page" to="154">154</biblScope><biblScope unit="page-count">18</biblScope><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="1998-03">1998-03</date></imprint><idno type="ISSN">0248-4900</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0248-4900</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Alkaline phosphatase</term><term>Amplification</term><term>Amplification mixture</term><term>Amplification products</term><term>Amplification reaction</term><term>Annealing</term><term>Appropriate controls</term><term>Avian myeloblastosis virus</term><term>Bagasra</term><term>Biological material</term><term>Boca york</term><term>Boehringer mannheim</term><term>Careful optimization</term><term>Cdna</term><term>Cell membrane</term><term>Cell suspensions</term><term>Cycle number</term><term>Denaturation</term><term>Detection system</term><term>Diffusion artifacts</term><term>Digestion</term><term>Digestion step</term><term>Direct reaction</term><term>Dnase</term><term>Dnase digestion</term><term>Dntps</term><term>Electron microscopy</term><term>Elongation step</term><term>Embedding</term><term>Endocrine cell populations</term><term>Endogenous biotin</term><term>Ethanol</term><term>Extension time</term><term>Final concentration</term><term>First step</term><term>Fixation</term><term>Fixation time</term><term>Fixative</term><term>Gene expression</term><term>Gene rearrangements</term><term>Genome</term><term>Glass slides</term><term>Gold particles</term><term>Good preservation</term><term>Growth hormone receptor mrna</term><term>Haase</term><term>High number</term><term>Higher concentration</term><term>Host cell</term><term>Human papillomavirus</term><term>Human press</term><term>Humid chamber</term><term>Hybridization</term><term>Hybridization step</term><term>Hybridization temperature</term><term>Hydrophylic resin</term><term>Indirect reactions</term><term>Individual cells</term><term>Infectious agents</term><term>Komminoth</term><term>Light microscopy</term><term>Localization</term><term>Major pitfall</term><term>Mertani</term><term>Mispriming</term><term>Molecular biology</term><term>Molecular diagnosis</term><term>Morel</term><term>Mrna</term><term>Mrna coding</term><term>Negative strand</term><term>Nucleic</term><term>Nucleic acid</term><term>Nucleic acid sequences</term><term>Nucleic acid target</term><term>Nucleic acids</term><term>Nucleotide</term><term>Nuovo</term><term>Paraformaldehyde</term><term>Phosphate buffer</term><term>Pituitary gland section</term><term>Polymerase</term><term>Polymerase activity</term><term>Polymerase chain reaction</term><term>Positive cells</term><term>Positive strand</term><term>Possible adsorption</term><term>Possible degradation</term><term>Powerful technique</term><term>Primer</term><term>Proteolytic digestion</term><term>Protocol</term><term>Raven press</term><term>Receptor</term><term>Replication</term><term>Rnase</term><term>Ronsin</term><term>Room temperature</term><term>Second step</term><term>Secondary structures</term><term>Several approaches</term><term>Signal density</term><term>Target sequence</term><term>Tissue preparation</term><term>Tissue section</term><term>Tissue sections</term><term>Total volume</term><term>Transcriptase</term><term>Ultrathin</term><term>Ultrathin sections</term><term>Unlabeled nucleotides</term><term>Unlabeled probe</term><term>Vibratome sections</term><term>Viral</term><term>Viral genome</term><term>Viral methods</term><term>Visna virus</term><term>White resin</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell‐localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT‐PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl2, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT‐PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.</div></front></TEI><affiliations><list><country><li>France</li><li>Singapour</li></country><region><li>Auvergne-Rhône-Alpes</li><li>Rhône-Alpes</li></region><settlement><li>Lyon</li></settlement><orgName><li>Université nationale de Singapour</li></orgName></list><tree><country name="France"><noRegion><name sortKey="Morel, Gerard" sort="Morel, Gerard" uniqKey="Morel G" first="Gérard" last="Morel">Gérard Morel</name></noRegion><name sortKey="Berger, Martina" sort="Berger, Martina" uniqKey="Berger M" first="Martina" last="Berger">Martina Berger</name><name sortKey="Recher, Sophie" sort="Recher, Sophie" uniqKey="Recher S" first="Sophie" last="Recher">Sophie Recher</name><name sortKey="Ricard Lum, Sylvie" sort="Ricard Lum, Sylvie" uniqKey="Ricard Lum S" first="Sylvie" last="Ricard-Blum">Sylvie Ricard-Blum</name><name sortKey="Ronsin, Brice" sort="Ronsin, Brice" uniqKey="Ronsin B" first="Brice" last="Ronsin">Brice Ronsin</name></country><country name="Singapour"><noRegion><name sortKey="Mertani, Hichem C" sort="Mertani, Hichem C" uniqKey="Mertani H" first="Hichem C." last="Mertani">Hichem C. Mertani</name></noRegion><name sortKey="Lobie, Peter E" sort="Lobie, Peter E" uniqKey="Lobie P" first="Peter E." last="Lobie">Peter E. Lobie</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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