Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction.
Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle's performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3928441?pdf=render |
| _version_ | 1819106358309421056 |
|---|---|
| author | Walid Nachar David Busseuil Yanfen Shi Teodora Mihalache-Avram Mélanie Mecteau Eric Rhéaume Jean-Claude Tardif |
| author_facet | Walid Nachar David Busseuil Yanfen Shi Teodora Mihalache-Avram Mélanie Mecteau Eric Rhéaume Jean-Claude Tardif |
| author_sort | Walid Nachar |
| collection | DOAJ |
| description | Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle's performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n = 7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n = 11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD. |
| first_indexed | 2024-12-22T02:36:52Z |
| format | Article |
| id | doaj.art-7776aa3dafc74206a03f2e9133ea75de |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T02:36:52Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-7776aa3dafc74206a03f2e9133ea75de2022-12-21T18:41:45ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0192e8933110.1371/journal.pone.0089331Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction.Walid NacharDavid BusseuilYanfen ShiTeodora Mihalache-AvramMélanie MecteauEric RhéaumeJean-Claude TardifLeft ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle's performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n = 7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n = 11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.http://europepmc.org/articles/PMC3928441?pdf=render |
| spellingShingle | Walid Nachar David Busseuil Yanfen Shi Teodora Mihalache-Avram Mélanie Mecteau Eric Rhéaume Jean-Claude Tardif Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. PLoS ONE |
| title | Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. |
| title_full | Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. |
| title_fullStr | Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. |
| title_full_unstemmed | Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. |
| title_short | Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction. |
| title_sort | optimisation of reference genes for gene expression analysis in a rabbit model of left ventricular diastolic dysfunction |
| url | http://europepmc.org/articles/PMC3928441?pdf=render |
| work_keys_str_mv | AT walidnachar optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT davidbusseuil optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT yanfenshi optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT teodoramihalacheavram optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT melaniemecteau optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT ericrheaume optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction AT jeanclaudetardif optimisationofreferencegenesforgeneexpressionanalysisinarabbitmodelofleftventriculardiastolicdysfunction |