Abstract
BackgroundHeart 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.ResultsWe 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.ConclusionAccurate 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.
Highlights
Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease
In Australia and New Zealand, less than 50% of lungs offered for donation post brain stem death (BSD) are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs [1]
The aim of this study was to develop a set of reference genes that can be used for normalisation of expression data in the pulmonary artery and lung of BSD sheep
Summary
Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. 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. Lung transplantation represents the only prospect of improved survival and quality of life for patients with end stage pulmonary disease. Brain stem death (BSD) is a prerequisite for the majority of heart and lung transplantation, yet this process adversely affects organ function, with lung being the most adversely affected. A clinically relevant model of BSD is an important key towards the understanding of the lung dysfunction post BSD, and we have subsequently developed a novel, clinically relevant 24 hour ovine model. The ovine models are treated in an animal ICU setting, with similar electrolyte management and hormonal resuscitation (methylprednisolone, tri-iodothyrosine (T3) and vasopressin) to mimic treatment given to human lung transplant donors prior to transplantation
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