Selection of Reference Genes for qRT-PCR Normalization to study Hif1α and Hif2α Expression in Hypobaric Hypoxia Susceptible and Tolerant rats lung

Acute hypobaric hypoxia may damage brain and lung tissues due to the development of disorders such as High Altitude Cerebral Edema (HACE) and High Altitude Pulmonary Edema (HAPE). Rats, like humans, exhibit susceptibility and tolerance in the same population to such extreme conditions. We selected reference genes to normalize the qRT-PCR study in order to evaluate whether a change in expression of Hif1α and Hif2α occurs in lungs of such rats exposed to acute hypobaric hypoxia. The hypobaric hypoxia susceptible (HHS) and tolerant (HHT) Sprague-Dawley rat groups, formed on the basis of their gasping time, were exposed to a simulated altitude of 9144 m at 24°C in a decompression chamber for a short duration of one hour. A set of reference genes including Gapdh, Actb, Rpl11, Rpl10a, Rps15 and Ppia was examined for normalization in qRT-PCR study to analyze expression of target genes Hif1α and Hif2α in the lungs of these groups as compared to the normoxic control. Rpl11, Actb and Rps15 genes in combination represented the most suitable reference genes based upon GeNorm, NormFinder and BestKeeper analyses. Expression of Hif1α and Hif2α genes was reduced in both HHS and HHT rat lungs. However, enhanced protein expression of HIF-1 in HHS group, and weak expression of HIF-2 protein in both groups was recorded. Our findings suggest that HIF-1 may play a significant role in mediating early responses towards acute hypobaric hypoxia in lung samples of HHS as well as HHT rats, with its profound protein expression in the former.