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Laser-capture microdissection RNA-sequencing for spatial and temporal tissue-specific gene expression analysis in plants
journal contributionposted on 12.01.2021, 05:45 by Lim LiewLim Liew, Yan WangYan Wang, Marta Peirats-LlobetMarta Peirats-Llobet, Oliver BerkowitzOliver Berkowitz, James WhelanJames Whelan, Mathew LewseyMathew Lewsey
© 2020 JoVE Journal of Visualized Experiments. The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.