Technical brief: isolation of total DNA from postmortem human eye tissues and quality comparison between iris and retina.

ABSTRACT: Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies.DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene.Regarding concentration, the retina yielded 936 ng/?l of DNA, while the iris yielded 78 ng/?l of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/?l/mg vs. 7.42 ng/?l/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies.The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye tissues. Retinal tissue provides DNA of excellent quantity and quality for genotyping and downstream genomic studies. However, DNA isolated from iris tissues, and treated with bovine serum albumin, may also be a valuable source of DNA for genotyping and genomic studies.

SUBMITTER: Wang JC 

PROVIDER: S-EPMC3534137 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature