For analysis of SDF-1, C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) mRNA levels in RPE/choroid and for analysis of ocular marker (MITF, Pax-6, Otx, and Six-3) mRNA levels in transmigrated Sca-1+ cells in the chemotaxis assay and in PBMNCs, total mRNA was isolated from the respective sources (RNeasy Mini Kit; Qiagen Inc., Valencia, CA). mRNA was then reverse-transcribed (TaqMan Reverse Transcription Reagents; Applied Biosystems [ABI], Foster City, CA). Detection of SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, Six-3, and β-microglobulin mRNA levels was performed by real-time RT-PCR (ABI PRISM 7000 Sequence Detection System; ABI). A 25-μL reaction mixture contained 12.5 μL mix (SYBR Green PCR Master Mix), 10 ng cDNA template, 5′-CGT GAG GCC AGG GAA GAG T-3′ forward and 5′-TGA TGA GCA TGG TGG GTT GA-3′ reverse primers for SDF-1, 5′-CCA GCC GGG GAC CTC ACT TGT AG-3′ forward and 5′-GGT CTT CCT GCC GTT TCT CTG TTG-3′ reverse primers for C3, 5′-CCA GAT CCT TGT GGC TCC TAT C-3′ forward and 5′-GGT TAG CGA TTC AGT TCC GTG-3′ reverse primers for HGF, 5′-TTC CCA TCA CCC CTG TAA ATG-3′ forward and 5′-TTG GAG TAC TTG GTC TAG TTC TTA G-3′ reverse primers for LIF, 5′-GGA CTT TCC CTT ATC CCA TTC A-3′ forward and 5′-GTT CTT GCT TGA TGA TCC GAT TC-3′ reverse primers for MITF, 5′-GCA ACC TGG CTA GCG AAA AG-3′ forward and 5′-CCC GTT CAA CAT CCT TAG TTT ATC AT-3′ reverse primers for Pax-6, 5′-CCA ATT TGG GCC GAC TTT G-3′ forward and 5′-GCG TAA GGC GGT TGC TTT AG-3′ reverse primers for Otx, 5′-GAC ACG CCA CAG ACC AAT AGA A-3′ forward and 5′-ATC TCC CCT AAA TCC ACA GTG AGT-3′ reverse primers for Six-3, and 5′-CAT ACG CCT GCA GAG TTA AGC A-3′ forward and 5′-GAT CAC ATG TCT CGA TCC CAG TAG-3′ reverse primers for β-microglobulin. Primers were designed with primer express software. The threshold cycle (Ct), that is, the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was subsequently determined. Relative quantitation of SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, and Six-3 mRNA expression was calculated with the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control β-microglobulin gene and relative to a calibrator, is expressed as 2−ΔΔCt (fold difference), where ΔCt = Ct of target genes (SDF-1, C3, HGF, LIF, MITF, Pax-6, Otx, Six-3) –Ct of endogenous control gene (β-microglobulin), and ΔΔCt = ΔCt of samples for target gene −ΔCt of calibrator for the target gene.