RNA Extraction and RT-qPCR Analysis in ARPE19 Cells

We extracted RNA from the ARPE19 cell line using a TRI reagent® (MRC, Cincinnati, OH, USA) and an Econospin column for RNA (GeneDesign, Osaka, Japan). The columns were washed with Buffer RPE (Qiagen, Hilden, Netherlands) and Buffer RWT (Qiagen, Hilden, Netherlands). RNA was reverse-transcribed into cDNA using ReverTra Ace® qPCR RT Master Mix with gDNA remover (TOYOBO, Osaka, Japan) [21 (link)]. Real-time-PCR was performed using THUNDERBIRD® SYBR® qPCR Mix (TOYOBO, Osaka, Japan) with a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative amplification of cDNA fragments was calculated using the 2^−ΔΔCt method. Real-time PCR primer sequences were as follows: Vegf forward: TCTACCTCCACCATGCCAAGT, Vegf reverse: GATGATTCTGCCCTCCTCCTT, Glut1 forward: CGGGCCAAGAGTGTGCTAAA, Glut1 reverse: TGACGATACCGGAGCCAATG, Pdk1 forward: ACAAGGAGAGCTTCGGGGTGGATC, Pdk1 reverse: CCACGTCGCAGTTTGGATTTATGC, Bnip3 forward: GGACAGAGTAGTTCCAGAGGCAGTTC, Bnip3 reverse: GGTGTGCATTTCCACATCAAACAT, Gapdh forward: TCCCTGAGCTGAACGGGAAG, and Gapdh reverse, GGAGGAGTGGGTGTCGCTGT.