We examined 18 single-nucleotide polymorphisms (SNPs) in intron 1 of the RORA gene. Seven of these SNPs (rs12916023, rs730754, rs8034864, rs12900948, rs12591914, rs17237514, and rs4335725) were shown to be significant, either individually or as part of a haplotype, in a previous study of 150 sibling pairs who were extremely discordant for AMD; that is, the index patient with the neovascular form of AMD who had an unaffected sibling with normal maculae who was older than 65 years (detailed information on the EDSP cohort is described elsewhere4,23). These 7 significant SNPs were derived from an initial group of 148 SNPs chosen approximately every 3000 to 5000 base pairs (bp) to represent variation within the 730 kilobases of the RORA gene.5 For this analysis, we chose 11 additional tagging SNPs that surrounded the region encompassing the 7 significant SNPs using the HapMap (www.hapmap.org). In choosing the tagging SNPs, each SNP must have had (1) a minor allele frequency of 10% or greater, and (2) an r2 value of at least 0.8. We extracted DNA from the buffy coat fraction of centrifuged blood specimens using the QIAmp Blood Kit (Qiagen, Valencia, California). Sequenom SpectroDESIGNER software (version 18.104.22.168) (Sequenom, San Diego, California) was used to design Multiplex PCR (polymerase chain reaction) assays using a sequence that contained the SNP site and 100 bp of flanking sequence on either side of the SNP. Briefly, 10 ng of genomic DNA was amplified in a 5-uL reaction containing 1X HotStar Taq PCR buffer (Qiagen), 1.625 mM MgCl2, 500 uM each of dNTP (deoxyribonucleotide triphosphate), 100 nM each of PCR primer, and 0.5 U of HotStar Taq (Qiagen). The reaction was incubated at 94°C for 15 minutes followed by 45 cycles of 94°C for 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, and 3 minutes at 72°C. Excess dNTPs were then removed from the reaction by incubation with 0.3 U shrimp alkaline phosphatase (USB, Cleveland, Ohio) at 37°C for 40 minutes followed by 5 minutes at 85°C to deactivate the enzyme. Single-primer extension over the SNP was carried out in a final concentration of between 0.625 uM and 1.5 uM for each extension primer (depending on the mass of the probe), iPLEX termination mix (Sequenom, San Diego, California), and 1.35 U iPLEX enzyme (Sequenom) and cycled using a 2-step 200–short cycles program: 94°C for 30 seconds followed by 40 cycles of 94°C for 5 seconds, 5 cycles of 52°C for 5 seconds, 80°C for 5 seconds, and 72°C for 3 minutes. The reaction was then desalted by addition of 6 mg cation exchange resin followed by mixing and centrifugation to settle the contents of the tube. The extension product was then spotted onto a 384-well spectroCHIP before being flown in a matrix-assisted laser desorption/ionization/time-of-flight mass spectrometer. Data were collected during real time, using SpectroTYPER Analyzer 22.214.171.124, SpectraAQUIRE 126.96.36.199, and SpectroCALLER 188.8.131.52 (Sequenom). Genotypes for each subject were also checked manually as an additional quality control measure. All laboratory personnel were blinded to case/control status.