Retinas were homogenized in lysis buffer, and total RNA was extracted (RNeasy; Qiagen, Studio City, California). A kit (DNA-free, Applied Biosystems) was used to remove DNA contamination, and RNA quantity was determined. One microgram of RNA was reverse transcribed using a kit (High-Capacity cDNA Reverse Transcription, Applied Biosystems). The VEGF164 was chosen because it is the most prevalent of VEGF isoforms associated with intravitreous neovascularization in the 50/10 OIR model, is upregulated by repeated oxygen fluctuations,10 and is a ligand for NP1, which acts as a coreceptor with VEGFR2.1 Primers for the following were made by the University of North Carolina's Oligonucleotide Synthesis Core Facility (http://www.med.unc.edu/olioli/index.htm): VEGF164 (5′GCACATAGGAGAGATGAGCT 3′, 5′GCTCACAGTGATTTT CTGGC3′, Fam TGCAG CATAGCAGATGTGAATGCAGACC Tamara), VEGFR1 (5′CCACCTCCATGTTTGAAGAC3′, 5′AGTCCAGGTGAATCGCTTCA3′, Fam TACCAGCAGTCTGCTGACCTCCCC Tamara), VEGFR2 (5′CTCCATCTTTTGGTGGGATG3′, 5′GCTGGTCTGGTTGGAGCCT3′, Fam AGGCCACAGACTCCCTGCTTTTACTG3 Tamara), PEDF (5′CCAACTCTTTGCAGGACATG3′, 5′TCACAGGTTTGCCGGTAATC3′, Fam ACAGTCCTTGTTTGAGTCCCCTGAC Tamara), NP1 (5′GGAGCTACTGGGCTGTGAAG3′, 5′ATGTGGGGAAGTCTGA3′, Fam CACCCAATGGGAACCTA Tamara), NP2 (5′ACACAAGGAGCCATTTCCAG3′, 5′CGGATCCTGATGAAACGAGT3′, Fam CAAAGGGGGAAGATTGGATG Tamara), and PEDF receptor (PEDFR) (5′CCAACTCTTTGCAGGACATC3′, 5′TCACAGGTTTGCCGGTAATC3′, Fam ACAGTCCTTGTTTGAGTCCCCTGAC Tamara).25 Complementary DNA was mixed 1:1 with a master mix (Taqman Universal Master Mix, Applied Biosystems) and primers. Triplicate samples were cycled in a polymerase chain reaction (PCR) system (AB 7500, Applied Biosystems). The system's software calculates cycle threshold automatically for each well. Rat β-actin, which we found was stably expressed in retinas under these oxygen stresses, was used as the control gene and was amplified with forward and reverse primers TGCCTGACGGTCAGGTCA and CAGGAAGGAAGGCTGGAAG, respectively. Duplicate reactions with a total volume of 16 μL were run for each sample and control using the PCR system, and again the cycle threshold was automatically calculated for each well. Cycle threshold values were normalized to β-actin and were expressed as fold increase over the p0 value for each growth factor or receptor, enabling comparisons between RA and 50/10 OIR.