Stability of housekeeping gene expression in the rat retina during exposure to cyclic hyperoxia

Recent evidence suggests a genetic component to oxygen-induced retinopathy (OIR), a robust experimental model of human retinopathy of prematurity. OIR lends itself well to quantitative analysis of gene expression in rodents with well-defined genetic backgrounds. Such analysis by real-time reverse transcription polymerase chain reaction (RT-PCR) requires the use of reference genes as internal standards for purposes of normalization. We sought to identify housekeeping genes showing stable retinal expression across different rat strains and developmental stages, that were not regulated by oxygen tension. Real-time RT-PCR was used to examine in normal (control) neonatal rat retina the expression of five candidate reference genes: acidic ribosomal phosphoprotein (ARBP), cyclophilin A (CYCA), gamma 2 actin (ACTG2), hypoxanthine guanine phosphoribosyltransferase (HPRT), and RNA polymerase 2 (RNAP2). ACTG2 was poorly expressed, whereas quantification of CYCA was confounded by putative amplification of pseudogenes. Expression of ARBP, HPRT, and RNAP2 was then quantified in dissected retinas from neonatal rats of three inbred strains (Fischer 344, Sprague Dawley, and Dark Agouti) under two different conditions of exposure to inspired oxygen (exposure to room air for 14 days from birth; exposure to cyclic hyperoxia for 14 days from birth). The average variation in relative expression between each pair of these three genes within each of the six cDNA test samples was used to assess stability of gene expression, relative to a standard retinal cDNA pool. The relative expression values for ARBP and HPRT were more closely correlated (r2=0.80) than were those for either gene with RNAP2 (ARBP and RNAP2: r2=0.31; HPRT and RNAP2: r2=0.25). There was little variation among the six experimental groups for the normalized expression of ARBP or HPRT (p>0.05). In contrast, the normalized expression of RNAP2 varied significantly amongst experimental groups: Within each strain, expression was higher in the oxygen-exposed group than in the room air-exposed group (p<0.05). We conclude that ARBP and HPRT exhibit expression that is sufficiently stable under conditions of varying oxygen tension, to permit their use as housekeeping genes in at least one model of OIR in the neonatal rat.