Effects of hypoxia on mRNA expression of housekeeping genes in rat brain tissue and primary cultured neural cells

Abstract

Internal standards are critical for quantitative RNA analyses. Housekeeping genes are often used as internal standards with the assumption that their expression levels remain relatively constant in different experimental conditions. In this study, four commonly used housekeeping genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ?-actin, 28S rRNA and 18S rRNA were selected to test whether this assumption is tenable under hypoxic conditions. We tested the RNA expression level of these four genes in different hypoxic conditions. Rats subjected to acute hypoxia for 2 hours were used for tissue detection. Primary cultured neural stem cells from E13 fetal rats were treated with 3% O₂ or 10% O₂ for 24 hours for in vitro experiments. In both experiments, expression levels of 28S rRNA and 18S rRNA were constant, independent of hypoxia types. However, expression levels of GAPDH and ?-actin were all changed in all kinds of hypoxic conditions. In particular, the mRNA expression level of GAPDH was increased by 43.4% under 3% O₂ hypoxic conditions. These results suggest that 18S rRNA and 28S rRNA are reliable internal controls for comparative analyses of transcription under hypoxia. GAPDH appears particularly unfavorable for this purpose in hypoxic conditions.

Keywords

housekeeping genes / hypoxia / gene expression / standardization