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Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction.

Nachar W, Busseuil D, Shi Y, Mihalache-Avram T, Mecteau M, Rhéaume E, Tardif JC - PLoS ONE (2014)

Bottom Line: 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 is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.

View Article: PubMed Central - PubMed

Affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT
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.

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GeNorm and Normfinder ranking of the 10 reference genes tested for the mRNA expression stability in left ventricles of 18 rabbits (normal diet n = 7 and cholesterol diet n = 11).Sdha, Gapdh and Hprt1 are shown as the best 3 genes with the lowest geNorm M value (M =  0.161, M =  0.168 and M = 0.176, respectively). Lower M value corresponds to higher gene stability. However, the use of the Normfinder algorithm results in the selection of Hprt1 (0.049) as the most stable gene and suggests to use the average of Hprt1 and Rpl5 (0.042) as normalization factor.
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pone-0089331-g001: GeNorm and Normfinder ranking of the 10 reference genes tested for the mRNA expression stability in left ventricles of 18 rabbits (normal diet n = 7 and cholesterol diet n = 11).Sdha, Gapdh and Hprt1 are shown as the best 3 genes with the lowest geNorm M value (M =  0.161, M =  0.168 and M = 0.176, respectively). Lower M value corresponds to higher gene stability. However, the use of the Normfinder algorithm results in the selection of Hprt1 (0.049) as the most stable gene and suggests to use the average of Hprt1 and Rpl5 (0.042) as normalization factor.

Mentions: GeNorm analyses are based on pairwise correlation of the expression level for each gene with the rest of the tested genes. An average of expression stability, M value, is given for each gene. The expression of a gene with an M value < 0.5 is considered highly stable. We found that all of the 10 genes tested in the LVs are stably expressed as documented by their average expression stability below 0.5. Among them, the three genes with the lowest M values were Hprt1 (M = 0.176), Gapdh (M = 0.168) and Sdha (M = 0.161); see Figure 1 for gene ranking. Pairwise variation (V) analysis, which provides the optimal number of reference genes that should be used in normalization, was also calculated between NFn and NFn+1. The geometric mean of the three best genes (Sdha, Gapdh and Hprt1) is sufficient to calculate the normalization factor, as the geNorm V value obtained for the 2 to 3 best genes (V2-3 = 0.0575) is lower than 0.15 and inclusion of additional genes would not significantly decrease the variation of the normalization factor (Figure 2).


Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction.

Nachar W, Busseuil D, Shi Y, Mihalache-Avram T, Mecteau M, Rhéaume E, Tardif JC - PLoS ONE (2014)

GeNorm and Normfinder ranking of the 10 reference genes tested for the mRNA expression stability in left ventricles of 18 rabbits (normal diet n = 7 and cholesterol diet n = 11).Sdha, Gapdh and Hprt1 are shown as the best 3 genes with the lowest geNorm M value (M =  0.161, M =  0.168 and M = 0.176, respectively). Lower M value corresponds to higher gene stability. However, the use of the Normfinder algorithm results in the selection of Hprt1 (0.049) as the most stable gene and suggests to use the average of Hprt1 and Rpl5 (0.042) as normalization factor.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928441&req=5

pone-0089331-g001: GeNorm and Normfinder ranking of the 10 reference genes tested for the mRNA expression stability in left ventricles of 18 rabbits (normal diet n = 7 and cholesterol diet n = 11).Sdha, Gapdh and Hprt1 are shown as the best 3 genes with the lowest geNorm M value (M =  0.161, M =  0.168 and M = 0.176, respectively). Lower M value corresponds to higher gene stability. However, the use of the Normfinder algorithm results in the selection of Hprt1 (0.049) as the most stable gene and suggests to use the average of Hprt1 and Rpl5 (0.042) as normalization factor.
Mentions: GeNorm analyses are based on pairwise correlation of the expression level for each gene with the rest of the tested genes. An average of expression stability, M value, is given for each gene. The expression of a gene with an M value < 0.5 is considered highly stable. We found that all of the 10 genes tested in the LVs are stably expressed as documented by their average expression stability below 0.5. Among them, the three genes with the lowest M values were Hprt1 (M = 0.176), Gapdh (M = 0.168) and Sdha (M = 0.161); see Figure 1 for gene ranking. Pairwise variation (V) analysis, which provides the optimal number of reference genes that should be used in normalization, was also calculated between NFn and NFn+1. The geometric mean of the three best genes (Sdha, Gapdh and Hprt1) is sufficient to calculate the normalization factor, as the geNorm V value obtained for the 2 to 3 best genes (V2-3 = 0.0575) is lower than 0.15 and inclusion of additional genes would not significantly decrease the variation of the normalization factor (Figure 2).

Bottom Line: 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 is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.

View Article: PubMed Central - PubMed

Affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus