Dataset Information

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

ABSTRACT: Background:Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. Methods and results:We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. Conclusion:This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

SUBMITTER: Sakai K

PROVIDER: S-EPMC5797369 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

Sakai Kaori K Taconnat Ludivine L Borrega Nero N Yansouni Jennifer J Brunaud Véronique V Paysant-Le Roux Christine C Delannoy Etienne E Martin Magniette Marie-Laure ML Lepiniec Loïc L Faure Jean Denis JD Balzergue Sandrine S Dubreucq Bertrand B

Plant methods 20180203

<h4>Background</h4>Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo.<h4>Methods and results</h4>We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm< ...[more]

PMID: 29434651

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Project description:BackgroundThe recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required.ResultsIn this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras(v12) transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II).ConclusionsEpigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.

Dataset Information

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

Publications

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

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