Are reference genes, GAPDH, beta-Actin and H-beta-2-microgloblin affected under normoxic and hypoxic conditions in Hep3B cells?

Abstract

Abstract:The appropriate choice of an internal standard is criti-cal for quantitative RNA analyses. As housekeeping genes, glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH),b-Actin andH-b-2-Microgloblin are commonly employed as RNA internalstandards with the assumption that their expression levels remainrelatively constant in different experimental conditions. We havecompared the gene expression levels of this genes under normoxicand hypoxic conditions in Hep3B (Human hepatoma cell line)with 10% FCS and without FCS (0.1% BSA). The cells werepassaged (2 000 000 cells per flask) into 25 cm2in 5 ml medium.Chemical induced hypoxic conditions in Hep3B cells were createdwith 150lM final concentration of CoCl2 for different timeintervals, namely 24, 48 and 72 hours with 10% FCS and with-out FCS (0.1% BSA). In addition, the expression of HIF1a(Hypoxia Inducible Factor-1a) which is the indicator of hypoxicconditions were analyzed. To compare the expression of the indi-vidual gene examined, RT-PCR was performed using primersdesigned using published information on GAPDH,b-actin, H-b-2-Microgloblin and HIF-1amRNA sequences in GenBank.Hypoxia conditions were correlated with upregulation of thetranscription factor Hypoxia Inducible Factor-1a mRNA levels.Expression levels of H-b-2-Microgloblin RNA were constant,independent of O2 tension, but levels of GAPDH andb-Actinvaried widely with hypoxic conditions.