Preparation of planar retinal specimens: verification byhistology, mRNA profiling, and proteome analysis

[摘要] Purpose: Elucidation of the transcriptome and proteome of the normalretina will be difficult since it is comprised of at least 55 differentcell types. However the characteristic layered cellular anatomy of theretina makes it amenable to planar sectioning, enabling the generationof enriched retinal cell populations. The aim of this study was tovalidate a reproducible method for preparing enriched retinal layersfrom porcine retina.Methods: The thicknesses of the retinal photoreceptor, inner nuclearand ganglion cell, and fiber layers were determined by routine histologyof cross sections of fresh whole retina mounted on polyvinylidenedifluoride (PVDF) membrane. Dissected retina (5 mm2) was placed onPVDF membrane and a series of planar cryosections corresponding to thephotoreceptor and inner nuclear layer were removed leaving the ganglioncell and fiber layer which was subsequently detached from the membrane.The retinal specimens were stored at -80 °C. Representative planartissue sections were sonicated in ice-chilled 40 mM ammonium bicarbonatepH 7.9 and aliquots removed for RNA extraction. Quantitative RT-PCR wasused to analyze the mRNA expression of genes indicative of specificretinal layers. Ammonium bicarbonate protein extracts were centrifuged,lyophilized and prepared for direct liquid chromatography-tandem massspectrometry (LC-MS/MS) analysis using a Waters Q-Tof Ultima.Results: Histological analysis established the parameters for planarcryosectioning: photoreceptor layer (69±1.8 μm), outer plexiform(11±0.6 μm), inner nuclear layer (28±0.5 μm), innerplexiform, ganglion cell and fiber layer (100±5.3 μm). Geneexpression profiling provided an independent method for validating therespective retinal preparations. For example, glial fibrillary acidicprotein (GFAP) was expressed up to 21 fold higher in the inner retinal"ganglion cell enriched" fraction than in the outer retinal"photoreceptor enriched" fraction. The pattern was reversed for bluecone opsin, which was expressed up to 24 fold higher in the"photoreceptor enriched" fraction. Endogenous protein fragmentsindicative of each layer were identified by mass spectrometry and denovo sequence data obtained.Conclusions: Combined histological and mRNA expression profiling hasconfirmed the development of a reproducible method for generatingvalidated porcine retinal layers enriched for specific cell types.Direct proteome analysis detected endogenous peptide fragments ofcharacteristic retinal proteins. Further analysis of these enrichedretinal cell preparations will facilitate a more selective investigationof the retinal transcriptome and proteome than studies of the intactretina.

[学科分类] 生物化学/生物物理