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Selection of reference genes for normalisation of real-time RT-PCR in brain-stem death injury in Ovis aries.

Passmore M, Nataatmadja M, Fraser JF - BMC Mol. Biol. (2009)

Bottom Line: Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results.Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies.This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, The Prince Charles Hospital, Chermside, Queensland, Australia. M.Passmore@uq.edu.au

ABSTRACT

Background: Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD) is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD.

Results: We evaluated the expression stability of 6 candidate normalisation genes (ACTB, GAPDH, HGPRT, PGK1, PPIA and RPLP0) using real time quantitative PCR. There was a wide range of Ct-values within each tissue for pulmonary artery (15-24) and lung (16-25) but the expression pattern for each gene was similar across the two tissues. After geNorm analysis, ACTB and PPIA were shown to be the most stably expressed in the pulmonary artery and ACTB and PGK1 in the lung tissue of BSD sheep.

Conclusion: Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies. This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.

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Related in: MedlinePlus

Selection of candidate normalisation genes using geNorm analysis. Stepwise exclusion of the least stable genes by calculating the average expression stability value M in combined BSD and control samples for pulmonary artery (A) and lung (B) and separate gene analysis for BSD pulmonary artery (C), control pulmonary artery (D), BSD lung (E) and control lung (F). The x axis indicates the ranking of the genes from least to most stable. The average expression stability value M is calculated for each gene with the highest M value indicating the least stable gene.
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Figure 3: Selection of candidate normalisation genes using geNorm analysis. Stepwise exclusion of the least stable genes by calculating the average expression stability value M in combined BSD and control samples for pulmonary artery (A) and lung (B) and separate gene analysis for BSD pulmonary artery (C), control pulmonary artery (D), BSD lung (E) and control lung (F). The x axis indicates the ranking of the genes from least to most stable. The average expression stability value M is calculated for each gene with the highest M value indicating the least stable gene.

Mentions: The geNorm program was used to identify the most stably expressed reference genes [11]. This program ranks candidate reference genes according to a stability value M. This value represents the mean pairwise variation between a candidate reference gene and all the other studied genes. The lowest M value indicates genes with the most stable expression. Stepwise elimination of successive genes showed that PPIA and ACTB were the most stably expressed genes in sheep pulmonary artery and PGK1 and ACTB were the most stably expressed genes in sheep lung (Figure 3). Further analysis was performed separating out the controls from the pulmonary artery and lung. When analysed separately the optimal reference genes for control and BSD pulmonary artery, and control and BSD lung were identical. This shows the high stability of these reference genes and that there is tissue-associated variability of gene expression in BSD, underlining the importance of choosing the most stable normalisation genes.


Selection of reference genes for normalisation of real-time RT-PCR in brain-stem death injury in Ovis aries.

Passmore M, Nataatmadja M, Fraser JF - BMC Mol. Biol. (2009)

Selection of candidate normalisation genes using geNorm analysis. Stepwise exclusion of the least stable genes by calculating the average expression stability value M in combined BSD and control samples for pulmonary artery (A) and lung (B) and separate gene analysis for BSD pulmonary artery (C), control pulmonary artery (D), BSD lung (E) and control lung (F). The x axis indicates the ranking of the genes from least to most stable. The average expression stability value M is calculated for each gene with the highest M value indicating the least stable gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721835&req=5

Figure 3: Selection of candidate normalisation genes using geNorm analysis. Stepwise exclusion of the least stable genes by calculating the average expression stability value M in combined BSD and control samples for pulmonary artery (A) and lung (B) and separate gene analysis for BSD pulmonary artery (C), control pulmonary artery (D), BSD lung (E) and control lung (F). The x axis indicates the ranking of the genes from least to most stable. The average expression stability value M is calculated for each gene with the highest M value indicating the least stable gene.
Mentions: The geNorm program was used to identify the most stably expressed reference genes [11]. This program ranks candidate reference genes according to a stability value M. This value represents the mean pairwise variation between a candidate reference gene and all the other studied genes. The lowest M value indicates genes with the most stable expression. Stepwise elimination of successive genes showed that PPIA and ACTB were the most stably expressed genes in sheep pulmonary artery and PGK1 and ACTB were the most stably expressed genes in sheep lung (Figure 3). Further analysis was performed separating out the controls from the pulmonary artery and lung. When analysed separately the optimal reference genes for control and BSD pulmonary artery, and control and BSD lung were identical. This shows the high stability of these reference genes and that there is tissue-associated variability of gene expression in BSD, underlining the importance of choosing the most stable normalisation genes.

Bottom Line: Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results.Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies.This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, The Prince Charles Hospital, Chermside, Queensland, Australia. M.Passmore@uq.edu.au

ABSTRACT

Background: Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD) is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD.

Results: We evaluated the expression stability of 6 candidate normalisation genes (ACTB, GAPDH, HGPRT, PGK1, PPIA and RPLP0) using real time quantitative PCR. There was a wide range of Ct-values within each tissue for pulmonary artery (15-24) and lung (16-25) but the expression pattern for each gene was similar across the two tissues. After geNorm analysis, ACTB and PPIA were shown to be the most stably expressed in the pulmonary artery and ACTB and PGK1 in the lung tissue of BSD sheep.

Conclusion: Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies. This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.

Show MeSH
Related in: MedlinePlus