Autotaxin, LPA receptor and ECM RNA expression in Normal vs Glaucoma Lamina Cribrosa Cells

Lamina cribrosa (LC) cells from age-matched glaucoma and normal control patient donors were cultured. RNA was extracted from fresh LC cells by adding 1ml of TriReagent to cell culture flasks and transferring to a 1.5ml Eppendorf. 100μl chloroform was added and incubated on ice for 5 minutes before centrifuging at 12,000 RPM. 500μl isopropanol was added to the aqueous layer, incubated for 5 minutes on ice and centrifuged at 12,000 RPM. The supernatant was discarded. 1ml ethanol was added to the pellet, centrifuged, and discarded. The airdried pellet was resuspended in 10-20μl diethylpyrocarbonate treated water. RNA yield and quality was confirmed by nanodrop. 2-3 nanograms of RNA was converted to complimentary DNA by adding 1.5μl oligoDTs, and DEPC water to make a total volume 13μl. Samples were heated to 65°C for 5 minutes. 2μl deoxynucleotides were added to the samples, with 1μl RevertAid Reverse Transcriptase and 4μl 5x Reverse Transcriptase Buffer. This total volume of 20μl was incubated in a thermocycler at 45°C for 60 minutes and 70°C for 10 minutes. 1μl cDNA was added to wells of a white 96 well PCR plate in triplicate. 5μl SYBR GREEN, 0.5μl forward primer, 0.5μl reverse primer and 3μl DEPC water were added to each sample. Primer sequences were designed using prior studies and the National Center for Biotechnology Information’s Primer-BLAST tool. Primers were acquired from Sigma-Aldrich. 18S was used as a housekeeping gene. The LightCycler® 480 Instrument II (Roche) was used, and standard PCR cycles were as follows: Denaturation at 95°C for 5 min, denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, elongation at 72°C for 20 seconds. This sequence was repeated for 45 cycles from the second denaturation step to amplify the product, followed by a melt curve of 95°C for 5 seconds and 65°C for 1 minute. Relative change in gene expression was calculated using the 2-DDCt equation. LC cells of the same passage from the three normal donors and the three glaucoma donors were used for each experiment, with technical triplicates for each donor.