DNA amplification from osmicated, plastic-embedded eye tissues

In the premolecular era, mammalian samples were embedded in epoxy resin blocks, such as Epon or Poly/Bed, for future evaluation by electron microscopy. However, use of these archival specimens for more modern mutation characterization studies can be challenging. The aim of this study was to determine if genomic DNA could be extracted from osmicated archival epoxy-embedded tissues to a quality suitable for short-amplicon PCR amplification.

We selected nine archived Epon, Araldite, or Poly/Bed embedded blocks of mammalian retinal and corneal tissue that were ~10 mm in length, embedded in the 1970s to 1990s, and had an extensive phenotypic description. Tissues were fixed in several combinations of glutaraldehyde and osmium before embedding. The blocks were shaved of excess resin, fragmented, and digested using an epoxy resin removal solution. The softened plastic was cut with a scalpel, washed, drained, and incubated at 56 °C overnight in a tissue lysis solution containing Proteinase K. Trizol was added to the samples, which were further mechanically homogenized. Chloroform was added, and the samples were centrifuged at 4 °C and 12,000 g. Upon phase separation, the upper clear phase was removed, 95% EtOH was added, the mix was filtered through a mini-genomic DNA extraction column and washed twice, and DNA was eluted with 10 mM Tris-HCL. Following final removal of phenol contamination using water-saturated ether, the purified DNA was quantified and used for PCR amplification.

The extraction success was tested by targeted PCR amplification using primers that produced amplicons 90 to 260 bp in length and targeted genes relevant for inherited eye studies (progressive rod-cone degeneration-PRCD; rhodopsin-RHO; glucuronidase beta-GUSB1), plus an additional control gene receptor accessory protein 1 (REEP1). All but one of the epoxy-embedded eye samples were successfully amplified. Sanger sequencing confirmed the gene identity of amplified products.

By identifying methods to extract DNA from osmicated epoxy-embedded mammalian eye tissues, our results provide a valuable resource for determining the genetic basis of inherited diseases and for retroactively confirming molecular diagnoses based on microscopic analysis.