Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia.

The appropriate choice of an internal standard is critical for quantitative RNA analyses. As housekeeping genes, GAPDH, beta-actin, cyclophilin, and 28S rRNA are commonly employed as RNA internal standards with the assumption that their expression levels remain relatively constant in different experimental conditions. We tested this assumption under hypoxia (1% O2, 24 hours) compared to normoxia (20% O2, 24 hours) and compared RNA levels of these 4 housekeeping genes head to head using ribonuclease protection assays. Four biologically diverse cell lines with respect to clonal origin, neoplastic transformation, and growth rates were used in the comparison. Expression levels of 28S rRNA were constant, independent of O2 tension, but levels of GAPDH, beta-actin, and cyclophilin varied widely with hypoxia. In particular, GAPDH mRNA expression was increased by 21.2-75.1% under hypoxic conditions. Increased GAPDH transcription in hypoxia was correlated in the cancer cell lines with upregulation of the transcription factor Hypoxia Inducible Factor-1alpha protein levels in identical experimental conditions. These results suggest that 28S rRNA is a reliable internal control for comparative analyses of transcription under hypoxia; GAPDH appears particularly unfavorable for this purpose either in hypoxia or other experimental conditions that upregulate HIF-1alpha.