A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.
The overall goal of this procedure is to isolate single cells for microarray based transcriptome analysis. This is accomplished by first dissecting the retina from the surrounding tissue and then dissociating it into single cells. The second step is to harvest single cells and deposit them into PCR tubes containing lysis buffer.
Then the cells are lysed and the CDNA is amplified using reverse transcription, followed by polymerase chain reaction. Ultimately, hybridization of the single cell CD NA on an A iMatrix microarray is used to reveal the mRNAs expressed in a single cell. The main advantage of profiling the gene expression in single cells versus using whole tissue approaches is that single cell isolation allows you to gain access to very small populations of cells that would comprise the entire tissue.
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