Preparation of planar retinal specimens: verification by histology, mRNA profiling, and proteome analysis.
OBJECTIVE
Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina.


METHODS
The thicknesses of the retinal photoreceptor, inner nuclear and ganglion cell, and fiber layers were determined by routine histology of cross sections of fresh whole retina mounted on polyvinylidene difluoride (PVDF) membrane. Dissected retina (5 mm2) was placed on PVDF membrane and a series of planar cryosections corresponding to the photoreceptor and inner nuclear layer were removed leaving the ganglion cell and fiber layer which was subsequently detached from the membrane. The retinal specimens were stored at -80 degrees C. Representative planar tissue sections were sonicated in ice-chilled 40 mM ammonium bicarbonate pH 7.9 and aliquots removed for RNA extraction. Quantitative RT-PCR was used to analyze the mRNA expression of genes indicative of specific retinal layers. Ammonium bicarbonate protein extracts were centrifuged, lyophilized and prepared for direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Waters Q-Tof Ultima.


RESULTS
Histological analysis established the parameters for planar cryosectioning: photoreceptor layer (69+/-1.8 microm), outer plexiform (11+/-0.6 microm), inner nuclear layer (28+/-0.5 microm), inner plexiform, ganglion cell and fiber layer (100+/-5.3 microm). Gene expression profiling provided an independent method for validating the respective retinal preparations. For example, glial fibrillary acidic protein (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 blue cone opsin, which was expressed up to 24 fold higher in the "photoreceptor enriched" fraction. Endogenous protein fragments indicative of each layer were identified by mass spectrometry and de novo sequence data obtained.


CONCLUSIONS
Combined histological and mRNA expression profiling has confirmed the development of a reproducible method for generating validated porcine retinal layers enriched for specific cell types. Direct proteome analysis detected endogenous peptide fragments of characteristic retinal proteins. Further analysis of these enriched retinal cell preparations will facilitate a more selective investigation of the retinal transcriptome and proteome than studies of the intact retina.
McKay
Gareth J
GJ
Ophthalmic Research Centre, Institute of Clinical Science, Queen's University of Belfast, Belfast, Northern Ireland.
Campbell
Lucinda
L
Oliver
Mike
M
Brockbank
Simon
S
Simpson
David A C
DA
Curry
William J
WJ
D019412:Anatomy, Cross-Sectional; D000818:Animals; D002853:Chromatography, Liquid; D016616:Cryoultramicrotomy; D005136:Eye Proteins / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D013058:Mass Spectrometry; D020411:Oligonucleotide Array Sequence Analysis; D010446:Peptide Fragments; D020543:Proteome / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling; D013552:Swine
D005136:Eye Proteins; D010446:Peptide Fragments; D020543:Proteome; D012333:RNA, Messenger