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Y. Han, S.C. Massey; Gene Expression Profiling within a single mouse retina neuron . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4257.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It is well known that retina utilizes more than 50 distinct cell types in coding and initial analysis of visual information. Our knowledge on functional roles of different retinal neurons is, however, still very limited because of the limitations of traditional analytic techniques. The objective of this study is to establish a new approach for classifying neurons by combining single–cell RT–PCR with anatomical and electrophysiological methods to confront the complexity of retinal circuits. Methods: Retinas from C57BL/6J mice between 4–8 weeks were used in this study. mRNA of single retinal neurons was collected either from retinal slice preparation by whole–cell patch clamp technique or from dissociated solitary retinal neurons by cell picking with glass electrodes. After converting from mRNA using oligo dT primer and AMV/MMLV reverse transcriptase, the first strand cDNA was tailed with dATP and unbiased amplified by PCR procedure. The amplified single cell cDNA was then used as template for PCR with gene–specific primers or was fragmented, labeled with Biotin–N6–ddATP to explore hybridization to a microarray genechip. Results: mRNA from photoreceptors (both rods and cones), bipolar cells (rod bipolar and on– and off–cone bipolar cells), müller cells, horizontal cells and amacrine cells have been collected. Single cell cDNAs were synthesized and amplified with good quality as judged by the expression of control genes. Cell specific marker genes (including rhodopsin, cone opsin, PKCα, mGluR6, retinaldehyde binding protein 1, calbindin–28) were detected only in specific retinal neurons as expected. Furthermore, we also explore applying the amplified single cell cDNA to an Affymetrix genechip to study gene expression profiles. Conclusions: The diverse repertory of transcripts contained in the identified individual retinal neurons can be amplified and multiple genes expression patterns can be studied with amplified single cell cDNA. The approach on gene expression profiling from single identified retinal neuron should further our understanding of functional organization of retina circuits and provide valuable insights into both physiological processes and various diseases states.
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