Measurement of feline cytokine gene expression by quantitative-competitive RT-PCR
Identifieur interne : 003A55 ( Main/Exploration ); précédent : 003A54; suivant : 003A56Measurement of feline cytokine gene expression by quantitative-competitive RT-PCR
Auteurs : Gregg A. Dean [États-Unis] ; Joanne Higgins [États-Unis] ; Alora Lavoy [États-Unis] ; Zhanyun Fan [États-Unis] ; Niels C. Pedersen [États-Unis]Source :
- Veterinary Immunology and Immunopathology [ 0165-2427 ] ; 1998.
English descriptors
- Teeft :
- Amplification, Amplification efficiency, Assay, Bamhirecori fragment, Band intensities, Cdna, Chain reaction, Competitive templates, Competitor, Competitor cdna, Competitor mrna, Competitor sequences, Consensus primers, Cytokine, Cytokine determination, Cytokine gene expression, Cytokine mrna, Cytokine primer sequence, Cytokine sequences, Elsevier science, Ethidium bromide, Feline, Feline cytokine gene expression, Feline cytokine sequences, Feline lymphocyte, Feline lymphocytes, Feline primers, Final primers, Gene expression, Greater size, Heteroduplex formation, Heterologous competitor, Immunology, Immunopathology, Large primers, Lymphocyte, Metaphor agarose, Metaphore agarose, Mrna, Native sequence, Native sequences, Native template, Plateau phase, Polymerase promoter site, Primer, Primer sequence, Quantitation, Rnase inhibitor, Single band, Southern blot analysis, Standard curve, Unknown samples, Veterinary immunology.
Abstract
Abstract: We have developed a method to quantitate feline cytokine gene expression using competitive RT-PCR. Feline cytokine specific primers were developed that encompass an intron, thus allowing differentiation of cDNA vs. genomic DNA amplification products. The PCR products of the primers were verified by sequencing and Southern blot analysis. For quantitation, a non-homologous RNA competitor was created for each cytokine of interest. The competitor was designed to yield an RT-PCR product 10–20% larger than the native sequence, thereby allowing differentiation of the two products by electrophoresis on an agarose gel. Both competitor and native sequences used the same primer sequences for RT (oligo dT) and PCR (cytokine specific). The amplification efficiency of the competitor and native sequence was shown to be identical which allowed comparison at any point during the amplification, including the plateau phase. The quantity of starting cytokine mRNA was determined by interpolation from a standard curve. As little as 1 μg of total cellular RNA was required per cytokine determination. The assay can routinely quantify as few as 1000 copies of template and spans a range of up to 4 log.
Url:
DOI: 10.1016/S0165-2427(98)00084-1
Affiliations:
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Pedersen</name><affiliation wicri:level="1"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616</wicri:regionArea><wicri:noRegion>CA 95616</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j">Veterinary Immunology and Immunopathology</title><title level="j" type="abbrev">VETIMM</title><idno type="ISSN">0165-2427</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">63</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="73">73</biblScope><biblScope unit="page" to="82">82</biblScope></imprint><idno type="ISSN">0165-2427</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0165-2427</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Amplification</term><term>Amplification efficiency</term><term>Assay</term><term>Bamhirecori fragment</term><term>Band intensities</term><term>Cdna</term><term>Chain reaction</term><term>Competitive templates</term><term>Competitor</term><term>Competitor cdna</term><term>Competitor mrna</term><term>Competitor sequences</term><term>Consensus primers</term><term>Cytokine</term><term>Cytokine determination</term><term>Cytokine gene expression</term><term>Cytokine mrna</term><term>Cytokine primer sequence</term><term>Cytokine sequences</term><term>Elsevier science</term><term>Ethidium bromide</term><term>Feline</term><term>Feline cytokine gene expression</term><term>Feline cytokine sequences</term><term>Feline lymphocyte</term><term>Feline lymphocytes</term><term>Feline primers</term><term>Final primers</term><term>Gene expression</term><term>Greater size</term><term>Heteroduplex formation</term><term>Heterologous competitor</term><term>Immunology</term><term>Immunopathology</term><term>Large primers</term><term>Lymphocyte</term><term>Metaphor agarose</term><term>Metaphore agarose</term><term>Mrna</term><term>Native sequence</term><term>Native sequences</term><term>Native template</term><term>Plateau phase</term><term>Polymerase promoter site</term><term>Primer</term><term>Primer sequence</term><term>Quantitation</term><term>Rnase inhibitor</term><term>Single band</term><term>Southern blot analysis</term><term>Standard curve</term><term>Unknown samples</term><term>Veterinary immunology</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: We have developed a method to quantitate feline cytokine gene expression using competitive RT-PCR. Feline cytokine specific primers were developed that encompass an intron, thus allowing differentiation of cDNA vs. genomic DNA amplification products. The PCR products of the primers were verified by sequencing and Southern blot analysis. For quantitation, a non-homologous RNA competitor was created for each cytokine of interest. The competitor was designed to yield an RT-PCR product 10–20% larger than the native sequence, thereby allowing differentiation of the two products by electrophoresis on an agarose gel. Both competitor and native sequences used the same primer sequences for RT (oligo dT) and PCR (cytokine specific). The amplification efficiency of the competitor and native sequence was shown to be identical which allowed comparison at any point during the amplification, including the plateau phase. The quantity of starting cytokine mRNA was determined by interpolation from a standard curve. As little as 1 μg of total cellular RNA was required per cytokine determination. The assay can routinely quantify as few as 1000 copies of template and spans a range of up to 4 log.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Dean, Gregg A" sort="Dean, Gregg A" uniqKey="Dean G" first="Gregg A" last="Dean">Gregg A. Dean</name></noRegion><name sortKey="Fan, Zhanyun" sort="Fan, Zhanyun" uniqKey="Fan Z" first="Zhanyun" last="Fan">Zhanyun Fan</name><name sortKey="Higgins, Joanne" sort="Higgins, Joanne" uniqKey="Higgins J" first="Joanne" last="Higgins">Joanne Higgins</name><name sortKey="Lavoy, Alora" sort="Lavoy, Alora" uniqKey="Lavoy A" first="Alora" last="Lavoy">Alora Lavoy</name><name sortKey="Pedersen, Niels C" sort="Pedersen, Niels C" uniqKey="Pedersen N" first="Niels C" last="Pedersen">Niels C. Pedersen</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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