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. 2018 Feb 3:14:10.
doi: 10.1186/s13007-018-0275-x. eCollection 2018.

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

Kaori Sakai #  1 , Ludivine Taconnat #  2   3 , Nero Borrega  1 , Jennifer Yansouni  2   3 , Véronique Brunaud  2   3 , Christine Paysant-Le Roux  2   3 , Etienne Delannoy  2   3 , Marie-Laure Martin Magniette  2   3   4 , Loïc Lepiniec  1 , Jean Denis Faure  1 , Sandrine Balzergue  2   3   5 , Bertrand Dubreucq  1
Affiliations

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

Kaori Sakai et al. Plant Methods. .

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.

Keywords: Arabidopsis; Embryo cells; Epidermis; Laser-assisted microdissection; Ultra-low-RNA-seq.

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Figures

Fig. 1
Fig. 1
Quantity and quality of extracted RNA related to microdissected surface coming from whole cotyledons embryo. a Relationship between RNA quality (RIN) and quantity extracted (pg). b Relationship between RNA quantity (pg) and the amount of microdissected surface (µm2). c Relationship between microdissected surface (µm2) and RNA quality (RIN)
Fig. 2
Fig. 2
Micro-dissected samples in this study. a Mesophyll or epiderm of linear staged embryos were microdissected at X40. Image shows the different steps of microdissection process: area selection, laser cutting, catapulting and capture of the sample. In red the epidermis, in blue the mesophyll. Bar = 30 µm. b Quality of the total RNA (Agilent Bioanalyzer profile) extracted after microdissection of the epidermis. 1: marker, 2: small RNA, 3: 18S rRNA, 4: 28S rRNA
Fig. 3
Fig. 3
Boxplot of normalized counts after Log2 + 1 transformation, for the 6 samples of the pilot experiment (RNA quantity from 5 ng to 10 pg)
Fig. 4
Fig. 4
Scatter-plot matrix of the normalized counts after Log2 + 1 transformation for the 6 samples of the pilot experiment (RNA quantity from 5 ng to 10 pg). The scatter-plot matrix shows histograms of the variables in the diagonal. Each cell on the bottom of the diagonal contains Spearman’s correlation coefficient between the pair of variables indicated in the diagonal. Each cell on the top of the diagonal corresponds to the plot of the pair of variables indicated in the diagonal
Fig. 5
Fig. 5
Genes involved in VLCFA and WAXES are up regulated in the epidermis. Schematic representation of the very long chain fatty acid and waxes biosynthetic pathway. Adapted from http://aralip.plantbiology.msu.edu/pathways/fatty_acid_elongation_wax_biosynthesis. The red dots highlight the differentially expressed genes
Fig. 6
Fig. 6
Genes involved in phenylpropanoids pathway are up regulated in the epidermis. Schematic representation of the phenylpropanoids pathway. Adapted from [43], New phytol, 202:132:144. EBG: early biosynthetic genes. LBG: late biosynthetic genes. The red dots highlight the differentially expressed genes
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