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SJ Lee, RK Shuler, BD Sippy, J Nickerson, J Boatright, HE Grossniklaus; Murine Retinal/RPE/Choroidal Expression of ABCA1, ACAT1 and ACAT2 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2607.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We recently demonstrated reliable production of basal laminar deposit (BlamD)-like material in C57BL/6 mice treated with argon blue laser and fed a high fat diet. As an initial step toward investigating a potential role of ABCA1, ACAT1 and ACAT2 in BlamD-like material formation, we attempted to determine if these three genes involved with lipid metabolism are expressed in the murine eye. Methods: Primers were designed for mouse genes ABCA1, ACAT1 and ACAT2 such that they are identical to nucleotides with exons and span at least one intron. Total RNA was extracted from the posterior poles (which included retina, RPE and choroid) of 3 month old 129S/v mouse eyes. Quantitative real time RT-PCR was performed. A short elongation time was used in order to favor synthesis of reverse-transcribed cDNA over genomic DNA templates. Melt curve analyses of the products were performed. The same primers were used in PCR reactions utilizing mouse genomic DNA as a template. Products from both amplification reactions were electrophoresed across 1% agrose gels. Results: Melt curve analysis and gel electrophoresis demonstrated a single product for each reaction. Real time RT-PCR generated the expected product lengths of 181, 162 and 152 bp. Genomic PCR product lengths were approximately 700, 2500 and 850 bp, respectively. The larger product for each genomic PCR reaction demonstrated the presence of at least one intron within each product. Product formation curves crossed threshold at 22.4, 18.7, and 32.2 cycles for ABCA1, ACAT1 and ACAT2, respectively. Conclusions: Quantitative real time RT-PCR measured RNA levels without DNA contamination. Expression of all three genes was present in the posterior pole of 129S/v mouse eyes. In the mouse posterior pole, ABCA1 appears to be expressed sixteen fold times less that ACAT1, while a much lower level of ACAT2 is expressed.
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