Absence of Beta-2 microgloblulin (B2M) and hypoxanthine-guanine phosphoribosyl transferase-1(HPRT1) gene modulation in U87MG and U251 Glioblastoma cell lines subjected to cobalt chloride mediated hypoxia

Hypoxia induces broad variations in expression levels of various housekeeping genes. Random selection of housekeeping genes as endogenous control in real time PCR based studies in hypoxia may lead to erroneous interpretation of results and hence such studies require careful selection of valid endogenous controls. In this context, we analyzed the suitability of four most commonly used housekeeping genes as endogenous controls in glioblastoma cell lines exposed to hypoxic conditions. Glioblastoma cell lines U87MG and U251 were subjected to normoxia and cobalt chloride induced hypoxia over varying dosages and time. Expression profiles of four housekeeping genes namely β-2 microglobulin (B2M), hypoxanthine-guanine phosphoribosyl transferase-1 (HPRT1), β-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analyzed by RT-qPCR. The results were statistically analyzed to select reference genes with stable expression profiles. The statistically analyzed results were validated by using NormFinder and BestKeeper softwares. Through this work we report that B2M and HPRT1 exhibit stable expression profile under hypoxic and normoxic experimental conditions. On the other hand, expression levels of GAPDH and ACTB were found to be significantly regulated by hypoxia. Thus B2M and HPRT1 should be considered as candidate endogenous controls for studies with similar experimental setting.