Abstract
BackgroundTibetans have lived at high altitudes for thousands of years, and they have unique physiological traits that enable them to tolerate this hypoxic environment. However, the genetic basis of these traits is still unknown. As a sensitive and highly efficient technique, RT-qPCR is widely used in gene expression analyses to provide insight into the molecular mechanisms underlying environmental changes. However, the quantitative analysis of gene expression in blood is limited by a shortage of stable reference genes for the normalization of mRNA levels. Thus, systematic approaches were used to identify potential reference genes.ResultsThe expression levels of eight candidate human reference genes (GAPDH, ACTB, 18S RNA, β2-MG, PPIA, RPL13A, TBP and SDHA) were assessed in blood from hypoxic environments. The expression stability of these selected reference genes was evaluated using the geNorm, NormFinder and BestKeeper programs. Interestingly, RPL13A was identified as the ideal reference gene for normalizing target gene expression in human blood before and after exposure to high-altitude conditions.ConclusionThese results indicate that different reference genes should be selected for the normalization of gene expression in blood from different environmental settings.
Highlights
Hypoxia is a major biological feature of high-altitude regions (Beall, 2000)
An increasing number of studies show that the hypoxia-inducible factor (HIF) signaling pathway plays a vital role in the adaptation to hypoxia (Ji et al, 2012)
To examine the stability of eight housekeeping gene (HKG) before and after migrating onto the plateau, the expression levels were evaluated by RT-qPCR, and the Shapiro–Wilk test was used to evaluate the normality of the Ct values (Table 3)
Summary
Hypoxia is a major biological feature of high-altitude regions (Beall, 2000). In hypoxic environments, transcription of various genes, such as endothelial PAS domain-containing protein 1 (EPAS1) and prolyl hydroxylase domain-containing protein 2 (PHD2), is initiated by hypoxia-related pathways. Tibetans have lived at high altitudes for thousands of years, and they have unique physiological traits that enable them to tolerate this hypoxic environment. The quantitative analysis of gene expression in blood is limited by a shortage of stable reference genes for the normalization of mRNA levels. The expression levels of eight candidate human reference genes (GAPDH, ACTB, 18S RNA, β2-MG, PPIA, RPL13A, TBP and SDHA) were assessed in blood from hypoxic environments. RPL13A was identified as the ideal reference gene for normalizing target gene expression in human blood before and after exposure to high-altitude conditions. These results indicate that different reference genes should be selected for the normalization of gene expression in blood from different environmental settings
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