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A simple method for gene expression and chromatin profiling of individual cell types within a tissue.

ABSTRACT: Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and show that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in the trimethylation of histone H3 at lysines 4 and 27. This method should be applicable to any organism that is amenable to transformation.

SUBMITTER: Deal RB

PROVIDER: S-EPMC2905389 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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A simple method for gene expression and chromatin profiling of individual cell types within a tissue.

Deal Roger B RB Henikoff Steven S

Developmental cell 20100601 6

Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure g ...[more]

PMID: 20627084

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Project description:BACKGROUND:An important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed. RESULTS:An optimised workflow for in-tube and on-slide in situ PCR is presented that has been evaluated using multiple plant species and tissue types. The protocol includes optimised methods for: (i) fixing, embedding, and sectioning of plant tissue; (ii) DNase treatment; (iii) in situ RT-PCR with the incorporation of DIG-labelled nucleotides; (iv) signal detection using colourimetric alkaline phosphatase substrates; and (v) mounting and microscopy. We also provide advice on troubleshooting and the limitations of using fluorescence as an alternative detection method. Using our protocol, reliable results can be obtained within two days from harvesting plant material. This method requires limited specialized equipment and can be adopted by any laboratory with a vibratome (vibrating blade microtome), a standard thermocycler, and a microscope. We show that the technique can be used to localise gene expression with cell-specific resolution. CONCLUSIONS:The in situ PCR method presented here is highly sensitive and specific. It reliably identifies the cellular expression pattern of even highly homologous and low abundance transcripts within target tissues, and can be completed within two days of harvesting tissue. As such, it has considerable advantages over other methods, especially in terms of time and cost. We recommend its adoption as the standard laboratory technique of choice for demonstrating the cellular expression pattern of a gene of interest.

Dataset Information

A simple method for gene expression and chromatin profiling of individual cell types within a tissue.

Publications

A simple method for gene expression and chromatin profiling of individual cell types within a tissue.

Similar Datasets

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