Tea Melinda, Michael Michael Zenon, Brereton Helen Mary, Williams Keryn Anne
Department of Ophthalmology, Flinders University of South Australia, Adelaide, Australia.
Mol Vis. 2013;19:501-8. Epub 2013 Feb 25.
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.
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.
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.
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.
氧诱导性视网膜病变（OIR）是一种成熟的人类早产儿视网膜病变动物模型，能够方便地研究视网膜基因和微小RNA（miRNA）表达随氧水平波动的变化。我们试图鉴定在暴露于不同氧水平、处于不同发育阶段以及在不同大鼠品系中均表现出稳定表达的小非编码RNA（ncRNA）基因，以作为在OIR大鼠模型中标准化miRNA表达的内参基因。
使用逆转录（RT）-qPCR在标准RNA库以及来自暴露于常氧和循环高氧环境的大鼠在出生后第5天（P5）、第6天（P6）、第9天（P9）和第14天的代表性视网膜样本上检测5种小ncRNA（U6小核RNA、miR-16、U87、4.5S RNA（H）“变体1”和5S核糖体RNA[rRNA]）的表达，分别评估发育阶段和暴露于氧水平波动的影响。使用两种近交白化大鼠品系，即Fischer 344（F344，对OIR有抗性）和Sprague-Dawley大鼠（SD，对OIR敏感），来评估大鼠品系对小ncRNA稳定性的影响。
在这个OIR大鼠模型中，5S rRNA的表达随品系、氧水平波动和发育阶段而变化。U6小核RNA在氧水平变化时稳定表达，在品系和发育阶段观察到的变化最小。与U6小核RNA相比，miR-16在氧水平变化和品系之间的表达稳定性较差。在发育阶段也观察到了一些表达变化。U6小核RNA和miR-16 TaqMan检测的PCR扩增效率分别为56%和78%。U87和4.5S RNA（H）“变体1”的表达随品系、暴露于循环高氧环境以及特别是发育阶段而变化，并且在新生大鼠视网膜中表达水平较低。
我们得出结论，U6小核RNA和miR-16是标准化miRNA表达最合适的参考RNA，因为它们在OIR大鼠模型中，相对于品系、暴露于循环高氧环境和发育阶段而言相对稳定。
