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Yan Guo, Steven L. Bernstein; Optic Nerve Gene Expression Analysis in a Rat Model of Nonarteritic Anterior Ischemic Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4615.
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Non-arteritic Anterior Ischemic Optic Neuropathy (NAION) is the leading cause of sudden optic nerve related vision loss. While the rodent anterior ischemic optic neuropathy (rAION) model can be used to evaluate many of the gene expression changes involved in NAION, a major limitation is the small amount of tissue (3mm X0.5mm) available from each individual, resulting in the use of many animals. Here we report the use of a novel linear isothermal RNA amplification method in the study of multiple gene expression in rAION.
25 male Sprague Dawley (180-230g) were used in the study. rAION was induced in left eye as previously described. The contralateral eye was utilized as untreated control. Eyes were examined one day post induction using slit lamp and/or optical coherence tomography (OCT). Animals were euthanized with CO2 at 5 different time points. The anterior 3mm of each optic nerve were harvested and snap frozen in liquid N2 until use. Total RNA was extracted, lyophilized following quality analysis by Agilient Bio-analyzer. 20ng of RNA from each optic nerve was pre-amplified using the WT-Ovation RNA amplification kit from NuGEN Technologies, Inc., CA. Real time PCR was performed using linear amplified, diluted cDNA with gene specific primers (aquaporins, GFAP, PTGD,cfos and the three NOS genes). 2 house keeping genes, β-actin and cyclophilin B, were used as internal controls. qPCR results were validated with the data obtained from traditionally generated cDNA via a Qiagen Ominiscript RT kit. The cycle threshold (dCt.) and difference of delta Ct. (ddCt.) were used in the statistical analyses using the Minitab 15 program.
We obtained reliable results (cycle number less than 30 cycles) with 1:1000 dilution of the amplified cDNA for medium abundant genes, and using 1:10 dilution for low abundant genes. The cycle number variance of the two house keeping genes is less than 1 cycle +/- 0.3 SD in 5 different samples. Individual gene ddCt’s from the amplified and non-amplified RNA were less than 1 in 80% of the genes we tested. There were consistent gene expression differences in samples from different groups.
The current pre-amplification method consistently amplifies total RNA with minimal tissue amounts and enables analysis of differential gene expression over a wide range of population abundance. In addition to its consistency and reproducibility, the amplification enables interrogation of many genes from a single rat optic nerve sample, which can dramatically reduce the number of animals needed in the study.
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