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 2 85 rRNA are commonly employed as RNA internal standards with the assumption that their expression levels remain relatively constant in different exper imental conditions. We tested this assumption under hypoxia (1% O-2, 24 hou rs) compared to normoxia (20% O-2, 24 hours) and compared RNA levels of the se 4 housekeeping genes head to head using ribonuclease protection assays, Four biologically diverse cell lines with respect to clonal origin, neoplas tic transformation, and growth rates were used in the comparison. Expressio n levels of 285 rRNA were constant, independent of O-2 tension, but levels of GAPDH, beta-actin, and cyclophilin varied widely with hypoxia, In partic ular, GAPDH mRNA expression was increased by 21.2-75.1% under hypoxic condi tions. Increased GAPDH transcription in hypoxia was correlated in the cance r cell lines with upregulation of the transcription factor Hypoxia Inducibl e Factor-la protein levels in identical experimental conditions. These resu lts suggest that 285 rRNA is a reliable internal control for comparative an alyses of transcription under hypoxia; GAPDH appears particularly unfavorab le for this purpose either in hypoxia or other experimental conditions that upregulate HIF-1 alpha.. (C) 1999 Academic Press.