Stability of small non-coding RNA reference gene expression in the rat retina during exposure to cyclic hyperoxia

PURPOSE: Oxygen-induced retinopathy (OIR) is a robust animal model of human retinopathy of prematurity that readily allows changes in retinal gene and microRNA (miRNA) expression in response to fluctuations in oxygen levels to be studied. We sought to identify small non-coding RNA (ncRNA) genes that showed stable expression upon exposure to varying levels of oxygen, with different developmental stages and in different rat strains, to act as reference genes for normalizing miRNA expression in a rat model of OIR. METHODS: Expression of five small ncRNAs (U6 snRNA, miR-16, U87, 4.5S RNA (H) “Variant 1”, and 5S ribosomal RNA [rRNA]) were tested on a standard RNA pool and representative retinal samples from P5, P6, P9, and P14 from room air– and cyclic hyperoxia–exposed rats using reverse transcription (RT)-qPCR, to assess the effect of developmental stage and exposure to fluctuations in oxygen levels, respectively. Two strains of inbred albino rats, Fischer 344 (F344, resistant to OIR) and Sprague-Dawley rats (SD, susceptible to OIR), were used to assess the effect of rat strain on the stability of the small ncRNAs. RESULTS: In this rat model of OIR, 5S rRNA expression was variable with strain, fluctuations in oxygen levels, and developmental stage. U6 snRNA was stably expressed with changes in oxygen levels, and minimal variation was observed with strain and developmental stage. MiR-16 showed less stable expression with changes in oxygen levels and between strains compared to U6 snRNA. Some variation in expression in response to developmental stage was also observed. The PCR amplification efficiencies of the U6 snRNA and miR-16 TaqMan assays were 56% and 78%, respectively. U87 and 4.5S RNA (H) “Variant 1” expression varied with strain, exposure to cyclic hyperoxia, and in particular developmental stage, and was at low levels in the neonatal rat retina. CONCLUSIONS: We conclude that U6 snRNA and miR-16 are the most suitable reference RNAs for normalizing miRNA expression, as they are relatively stable with strain, exposure to cyclic hyperoxia, and developmental stage in a rat model of OIR.