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Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

Identifieur interne : 000487 ( Pmc/Curation ); précédent : 000486; suivant : 000488

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

Auteurs : Lies Bogaert [Belgique] ; Mario Van Poucke [Belgique] ; Cindy De Baere [Belgique] ; Luc Peelman [Belgique] ; Frank Gasthuys [Belgique] ; Ann Martens [Belgique]

Source :

BMC Biotechnology [ 1472-6750 ] ; 2006.

RBID : PMC:1484482

Abstract

Background

Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses.

Results

In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids.

Conclusion

Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1484482

DOI: 10.1186/1472-6750-6-24
PubMed: 16643647
PubMed Central: 1484482

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<author><name sortKey="Bogaert, Lies" sort="Bogaert, Lies" uniqKey="Bogaert L" first="Lies" last="Bogaert">Lies Bogaert</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium</nlm:aff>
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<country xml:lang="fr">Belgique</country>
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<author><name sortKey="De Baere, Cindy" sort="De Baere, Cindy" uniqKey="De Baere C" first="Cindy" last="De Baere">Cindy De Baere</name>
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<author><name sortKey="Van Poucke, Mario" sort="Van Poucke, Mario" uniqKey="Van Poucke M" first="Mario" last="Van Poucke">Mario Van Poucke</name>
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<affiliation wicri:level="1"><nlm:aff id="I1">Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
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<author><name sortKey="Peelman, Luc" sort="Peelman, Luc" uniqKey="Peelman L" first="Luc" last="Peelman">Luc Peelman</name>
<affiliation wicri:level="1"><nlm:aff id="I2">Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University - UGent, Heidestraat 19, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University - UGent, Heidestraat 19, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
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<author><name sortKey="Gasthuys, Frank" sort="Gasthuys, Frank" uniqKey="Gasthuys F" first="Frank" last="Gasthuys">Frank Gasthuys</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
<wicri:regionArea>Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke</wicri:regionArea>
</affiliation>
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<author><name sortKey="Martens, Ann" sort="Martens, Ann" uniqKey="Martens A" first="Ann" last="Martens">Ann Martens</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium</nlm:aff>
<country xml:lang="fr">Belgique</country>
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<series><title level="j">BMC Biotechnology</title>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually <italic>GAPDH </italic>
or <italic>ACTB</italic>
, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses.</p>
</sec>
<sec><title>Results</title>
<p>In the present study the gene transcription levels of 6 commonly used reference genes (<italic>ACTB, B2M, HPRT1, UBB, TUBA1 </italic>
and <italic>RPL32</italic>
) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, <italic>TUBA1, ACTB </italic>
and <italic>UBB </italic>
were found to be most stable in normal skin and <italic>B2M, ACTB </italic>
and <italic>UBB </italic>
in equine sarcoids.</p>
</sec>
<sec><title>Conclusion</title>
<p>Based on these results, <italic>TUBA1, ACTB </italic>
and <italic>UBB</italic>
, respectively <italic>B2M, ACTB </italic>
and <italic>UBB </italic>
can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of <italic>UBB, ACTB </italic>
and <italic>B2M </italic>
can be recommended as a reliable and accurate normalisation factor.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
  <front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Biotechnol</journal-id>
<journal-title>BMC Biotechnology</journal-title>
<issn pub-type="epub">1472-6750</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
<publisher-loc>London</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">16643647</article-id>
<article-id pub-id-type="pmc">1484482</article-id>
<article-id pub-id-type="publisher-id">1472-6750-6-24</article-id>
<article-id pub-id-type="doi">10.1186/1472-6750-6-24</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Methodology Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids</article-title>
</title-group>
<contrib-group><contrib id="A1" contrib-type="author"><name><surname>Bogaert</surname>
<given-names>Lies</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>lies.bogaert@UGent.be</email>
</contrib>
<contrib id="A2" contrib-type="author"><name><surname>Van Poucke</surname>
<given-names>Mario</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>mario.vanpoucke@UGent.be</email>
</contrib>
<contrib id="A3" contrib-type="author"><name><surname>De Baere</surname>
<given-names>Cindy</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>cindy.debaere@UGent.be</email>
</contrib>
<contrib id="A4" contrib-type="author"><name><surname>Peelman</surname>
<given-names>Luc</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>luc.peelman@UGent.be</email>
</contrib>
<contrib id="A5" contrib-type="author"><name><surname>Gasthuys</surname>
<given-names>Frank</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>frank.gasthuys@UGent.be</email>
</contrib>
<contrib id="A6" corresp="yes" contrib-type="author"><name><surname>Martens</surname>
<given-names>Ann</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>ann.martens@UGent.be</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University - UGent, Salisburylaan 133, B-9820 Merelbeke, Belgium</aff>
<aff id="I2"><label>2</label>
Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University - UGent, Heidestraat 19, B-9820 Merelbeke, Belgium</aff>
<pub-date pub-type="collection"><year>2006</year>
</pub-date>
<pub-date pub-type="epub"><day>27</day>
<month>4</month>
<year>2006</year>
</pub-date>
<volume>6</volume>
<fpage>24</fpage>
<lpage>24</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6750/6/24"></ext-link>
<history><date date-type="received"><day>19</day>
<month>12</month>
<year>2005</year>
</date>
<date date-type="accepted"><day>27</day>
<month>4</month>
<year>2006</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2006 Bogaert et al; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2006</copyright-year>
<copyright-holder>Bogaert et al; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment> 
 Bogaert 
 Lies 
  
 lies.bogaert@UGent.be 
  
 Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids 
 2006BMC Biotechnology 6(1): 24-. (2006)1472-6750(2006)6:1<24>urn:ISSN:1472-6750</pmc-comment>
        </license>
</permissions>
<abstract><sec><title>Background</title>
<p>Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually <italic>GAPDH </italic>
or <italic>ACTB</italic>
, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses.</p>
</sec>
<sec><title>Results</title>
<p>In the present study the gene transcription levels of 6 commonly used reference genes (<italic>ACTB, B2M, HPRT1, UBB, TUBA1 </italic>
and <italic>RPL32</italic>
) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, <italic>TUBA1, ACTB </italic>
and <italic>UBB </italic>
were found to be most stable in normal skin and <italic>B2M, ACTB </italic>
and <italic>UBB </italic>
in equine sarcoids.</p>
</sec>
<sec><title>Conclusion</title>
<p>Based on these results, <italic>TUBA1, ACTB </italic>
and <italic>UBB</italic>
, respectively <italic>B2M, ACTB </italic>
and <italic>UBB </italic>
can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of <italic>UBB, ACTB </italic>
and <italic>B2M </italic>
can be recommended as a reliable and accurate normalisation factor.</p>
</sec>
</abstract>
</article-meta>
</front>
</pmc>
</record>

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Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

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