Abstract
Purpose: :
While age–related changes in retinal gene expression are known to occur in primate retina, there has been considerable variability in the isolation, report, and confirmation of true age–related retinal gene expression changes. We wanted to determine which age–related retinal gene expression changes are real, which method(s) yield the most valid, reproducible results, and to determine whether identified gene expression changes correspond to functional retinal changes.
Methods: :
We utilized panels of both human and old world (rhesus) retinal tissue, ranging from 16–85y/o (human), and 2.5–30y/o (monkey). A minimum of 10 samples/age (20 samples total/species) were utilized. Primary age–related isolate candidates were obtained from duplicate membrane array analysis screened with old and young human retinal cDNA probes, and confirmed with old and young monkey cDNA probes. For additional controls, we also generated a number of specific cDNA fragments from previously reported ‘age–related’ genes. Age–related cDNA candidates were evaluated by a number of methods, including northern, RQ–PCR and RNAse protection assay (RPA). To confirm gene–functional changes, we performed 2–D–In Gel (Protein Electrophoresis)–Differential Expression (2–DIGE).
Results: :
Most (>80% of ‘age–related’ changes identified by single species analysis were disproved by re–testing with RNA from two primate species. The majority of age–related gene changes are in the range of 1–3 fold, including (in humans) inflammation–based changes. Many RQ–PCR–based analyses were not re–confirmable by Northern and RPA results. 2–DIGE analysis revealed that few proteins change during aging, again typically within a 1–3 fold change
Conclusions: :
The number of true age–related retinal gene changes resulting in alterations in protein function, are relatively few, but in important pathways. This suggests that, rather than massive changes, true age–related gene changes are subtle, but are likely to play important roles in changing retinal function during normal aging. The majority of ‘age–associated’ gene expression change must be taken with skepticism, unless confirmed by multiple techniques, including, but not exclusively RQ–PCR.
Keywords: aging • gene/expression • proteomics