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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

Pairwise variation to determine the optimal number of normalisation genes. The optimal number of genes was determined separately for pulmonary artery (A) and lung (B) in combined BSD and control samples. A large pairwise variation V indicates that the added gene has a significant effect and should be included in the normalisation factor calculation. Based on the pairwise variation for pulmonary artery and lung addition of a third reference gene does not improve normalisation in either tissue.
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Figure 4: Pairwise variation to determine the optimal number of normalisation genes. The optimal number of genes was determined separately for pulmonary artery (A) and lung (B) in combined BSD and control samples. A large pairwise variation V indicates that the added gene has a significant effect and should be included in the normalisation factor calculation. Based on the pairwise variation for pulmonary artery and lung addition of a third reference gene does not improve normalisation in either tissue.

Mentions: GeNorm was also used to determine the optimal number of reference genes for normalisation. The software calculates the normalisation factor (NF) from at least two genes at which the variable V defines the pairwise variation between sequential NFs. This means that V2/3 shows the variation of the NF of two genes in relation to three genes. We used a cut-off value of 0.15, below which the addition of an extra gene will not significantly improve normalisation [11]. Based on this assumption the addition of a third gene is not required for either pulmonary artery or lung (Figure 4) where the V2/3 values are 0.149 and 0.104 respectively. In fact, the addition of a third reference gene actually increases the pairwise variation to 0.179 for pulmonary artery and 0.118 for lung.


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)

Pairwise variation to determine the optimal number of normalisation genes. The optimal number of genes was determined separately for pulmonary artery (A) and lung (B) in combined BSD and control samples. A large pairwise variation V indicates that the added gene has a significant effect and should be included in the normalisation factor calculation. Based on the pairwise variation for pulmonary artery and lung addition of a third reference gene does not improve normalisation in either tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Pairwise variation to determine the optimal number of normalisation genes. The optimal number of genes was determined separately for pulmonary artery (A) and lung (B) in combined BSD and control samples. A large pairwise variation V indicates that the added gene has a significant effect and should be included in the normalisation factor calculation. Based on the pairwise variation for pulmonary artery and lung addition of a third reference gene does not improve normalisation in either tissue.
Mentions: GeNorm was also used to determine the optimal number of reference genes for normalisation. The software calculates the normalisation factor (NF) from at least two genes at which the variable V defines the pairwise variation between sequential NFs. This means that V2/3 shows the variation of the NF of two genes in relation to three genes. We used a cut-off value of 0.15, below which the addition of an extra gene will not significantly improve normalisation [11]. Based on this assumption the addition of a third gene is not required for either pulmonary artery or lung (Figure 4) where the V2/3 values are 0.149 and 0.104 respectively. In fact, the addition of a third reference gene actually increases the pairwise variation to 0.179 for pulmonary artery and 0.118 for lung.

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