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

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.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0275-x) contains supplementary material, which is available to authorized users.

Genetic activity during early plant embryogenesis

“…Transcriptomic studies can now measure and compare transcriptomes in individual cell types [ 64 , 65 , 255 ]. Some examples include comparison of egg to zygote transcriptomes [ 256 ], apical compared with basal cell transcriptomes [ 257 ], determination of an embryo epidermal specific transcriptome [ 258 ], and other subregions of the seed/embryo [ 259 ].…”

Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development

“…directly within the seed, in a cell-specific manner. Recent developments combining INTACT or laserassisted micro-dissection with RNA-seq, ATAC-and CHIP-seq (Belmonte et al, 2013;Buenrostro et al, 2015;Chan et al, 2016;Kelsey et al, 2017;Libault et al, 2017;Magnani et al, 2017;Sakai et al, 2018;Sijacic et al, 2018) may provide more precise and quantitative data to draw a comprehensive and predictive model of the LAFL network.…”

Infectious plant diseases: etiology, current status, problems and prospects in plant protection

“…The most common methods of serological diagnosis include enzyme immunoassay, immunoblotting, dot-blot hybridization, immunochromatography [ 68 ], and serologically specific electron microscopy [ 69 ]. Methods based on DNA detection include fluorescence in situ hybridization [ 70 ], various polymerase chain reaction (PCR) techniques, including nested PCR, cooperative PCR, multiplex PCR, real-time PCR, and DNA fingerprinting. There are also RNA-based approaches: isothermal amplification of nucleic acids [ 71 ], the AmpliDet RNA real-time diagnostic system [ 72 ], and reverse-transcription PCR.…”