Abstract
BackgroundGenome-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 resultsWe 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.ConclusionThis 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.
Highlights
Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology
In order to optimize RNA quality taking into consideration the microdissection process itself, we first evaluated the quantity and quality of total RNA extracted from the entire embryo
To go further in the transcriptional regulations occurring in the epidermis, we focused on the transcription factors (TF) that are differentially expressed with a log ratio > 1 in the epidermis when compared to the mesophyll cell (Table 3)
Summary
Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. Sakai et al Plant Methods (2018) 14:10 approaches have been used including, the expression of reporter genes coupled to affinity purification system (e.g. INTACT, [10], fluorescent-activated cell sorting [2, 13, 35, 44], or manual dissection based on morphological characters). These techniques are limited in their application due to the need for protoplasting or genetic transformations with cell type-specific markers. We combined ultra-low-RNA-seq sequencing with laser microdissection for undertaking precise and comprehensive analyses of the epidermal versus mesophyll cell transcriptomes of the cotyledons, at the torpedo-shaped stage of the small Arabidopsis embryo
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