A commercially available zooblot containing 8 μg of EcoRI-digested genomic DNA isolated from various species (human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast) was purchased (BD Biosciences-Clontech, Palo Alto, CA). A 376-bp DNA probe corresponding to exon 6 of the human ELOVL4 gene was generated from a genomic DNA template by polymerase chain reaction (PCR), with primers 5′-GAAGATGCCGATGTTGTTAAAAG-3′ and 5′-GTCAACAACAGTTAAGGCCCA-3′. PCR cycling parameters used were as follows: 1 cycle of denaturation at 94°C for 3 minutes; 30 cycles of denaturation at 94°C for 30 seconds, primer annealing at 55°C for 30 seconds, primer extension at 72°C for 40 seconds; and 1 cycle of extension at 72°C for 3 minutes. A probe corresponding to ELOVL4 coding regions was also generated by total cDNA synthesis with a commercial kit (Invitrogen Canada, Inc., Burlington, Ontario, Canada) and 5 μg of human retina total RNA as template, followed by PCR using 100 ng total cDNA template with primers 5′-GTGTGGAAAATTGGCCTCTG-3′ and 5′-CATGGCTGTTTTTCCAGCTT-3′ to amplify a 743-bp cDNA fragment containing protein-encoding sequences found within exons 2 through 6. PCR cycling parameters used were as follows: 1 cycle of denaturation at 94°C for 3 minutes; 30 cycles of denaturation at 94°C for 1 minute, primer annealing at 45°C for 1 minute, primer extension at 72°C for 3 minutes; and 1 cycle of extension at 72°C for 7 minutes. Probes were purified with a gel extraction kit (QIAquick; Qiagen Inc., Mississauga, Ontario, Canada) and radiolabeled with [α32P]-dCTP (Amersham Biosciences, Baie d’Urfé, Quebec, Canada) by random primer labeling using a DNA labeling system (Multiprime; Amersham Biosciences). Hybridization of each probe to the zooblot was performed in hybridization solution (Hybrisol II; Serologicals Corp., Norcross, GA) at 42°C for 16 hours using 106 cpm of labeled, purified probes per milliliter of hybridization solution. The blot was then washed twice for 15 minutes in 2× sodium chloride/tri-sodium citrate buffer (SSC), once for 30 minutes in 2× SSC and 0.1% sodium dodecyl sulfate (SDS), and once for 10 minutes in 0.1% SSC with 0.1% SDS all at 42°C, followed by exposure to autoradiograph film (X-Omat; Eastman Kodak, Rochester, NY) at −70°C for 24 hours.