Laser Microdissection–Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA

Chandran, Divya; Scanlon, Michael J.; Ohtsu, Kazuhiro; Timmermans, Marja C.P.; Schnable, Patrick S.; Wildermuth, Mary C.

doi:10.1002/0471142727.mb25a03s112

Cited by 11 publications

(4 citation statements)

References 67 publications

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“…RNA isolation and amplification were performed according to Chandran et al . () using the PicoPure RNA Extraction Kit and TargetAmp 2‐round aRNA Amplification Kit (Epicentre/Illumina, Madison, WI, USA), respectively.…”

Section: Co2 Labeling and Autoradiographymentioning

confidence: 99%

“…Microtome sectioning (10 lm thick) and laser microdissection were performed according to Chandran et al (2015). For microdissection, PALM (P.A.L.M.…”

Section: Lcm and Rna Isolationmentioning

confidence: 99%

“…A total area of approximately 1.0 mm 2 of tissue was collected for each sample with three biological replicates. RNA isolation and amplification were performed according to Chandran et al (2015) using the PicoPure RNA Extraction Kit and TargetAmp 2round aRNA Amplification Kit (Epicentre/Illumina, Madison, WI, USA), respectively.…”

Section: Lcm and Rna Isolationmentioning

confidence: 99%

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Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development

Sui

Nie

et al. 2018

The Plant Journal

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The phloem of the Cucurbitaceae has long been a subject of interest due to its complex nature and the economic importance of the family. As in a limited number of other families, cucurbit phloem is bicollateral, i.e. with sieve tubes on both sides of the xylem. To date little is known about the specialized functions of the internal phloem (IP) and external phloem (EP). Here, a combination of microscopy, fluorescent dye transport analysis, micro-computed tomography, laser capture microdissection and RNA-sequencing (RNA-Seq) were used to study the functions of IP and EP in the vascular bundles (VBs) of cucumber fruit. There is one type of VB in the peduncle, but four in the fruit: peripheral (PeVB), main (MVB), carpel (CVB) and placental (PlVB). The VBs are bicollateral, except for the CVB and PlVB. Phloem mobile tracers and C applied to leaves are transported primarily in the EP, and to a lesser extent in the IP. RNA-Seq data indicate preferential gene transcription in the IP related to differentiation/development, hormone transport, RNA or protein modification/processing/transport, and nitrogen compound metabolism and transport. The EP preferentially expresses genes for stimulus/stress, defense, ion transport and secondary metabolite biosynthesis. The MVB phloem is preferentially involved in photoassimilate transport, unloading and long-distance signaling, while the PeVB plays a more substantial role in morphogenesis and/or development and defense response. CVB and PlVB transcripts are biased toward development of reproductive organs. These findings provide an integrated view of the differentiated structure and function of the vascular tissue in cucumber fruit.

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Section: Co2 Labeling and Autoradiographymentioning

confidence: 99%

“…Microtome sectioning (10 lm thick) and laser microdissection were performed according to Chandran et al (2015). For microdissection, PALM (P.A.L.M.…”

Section: Lcm and Rna Isolationmentioning

confidence: 99%

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Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development

Sui

Nie

et al. 2018

The Plant Journal

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“…LCM (Fig. D and UNIT 25A.3 Chandran et al., here) enables the isolation of single cells from tissue sections under a microscope. After visualization, a laser beam is focused on cells of interest, which fuses the cells onto a thin, transparent film.…”

Section: Tissue Dissociationmentioning

confidence: 99%

Introduction to Single‐Cell RNA Sequencing

Olsen

Baryawno

2018

CP Molecular Biology

127

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During the last decade, high-throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA-seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA-seq is typically performed in "bulk," and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells. Single-cell RNA-seq (scRNA-seq) represents an approach to overcome this problem. By isolating single cells, capturing their transcripts, and generating sequencing libraries in which the transcripts are mapped to individual cells, scRNA-seq allows assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution. Here, we present the most common scRNA-seq protocols in use today and the basics of data analysis and discuss factors that are important to consider before planning and designing an scRNA-seq project. © 2018 by John Wiley & Sons, Inc.

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Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti

Roux

Rodde

Moreau

et al. 2018

Methods in Molecular Biology

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Understanding the development of multicellular organisms requires the identification of regulators, notably transcription factors, and specific transcript populations associated with tissue differentiation. Laser capture microdissection (LCM) is one of the techniques that enable the analysis of distinct tissues or cells within an organ. Coupling this technique with RNA sequencing (RNAseq) makes it extremely powerful to obtain a genome-wide and dynamic view of gene expression. Moreover, RNA sequencing allows two or potentially more interacting organisms to be analyzed simultaneously. In this chapter, a LCM-RNAseq protocol optimized for root and symbiotic root nodule analysis is presented, using the model legume Medicago truncatula (in interaction with Sinorhizobium meliloti in the nodule samples). This includes the description of procedures for plant material fixation, embedding, and micro-dissection; it is followed by a presentation of techniques for RNA extraction and amplification, adapted for the simultaneous analysis of plant and bacterial cells in interaction or, more generally, polyadenylated and non-polyadenylated RNAs. Finally, step-by-step statistical analyses of RNAseq data are described. Those are critical for quality assessment of the whole procedure and for the identification of differentially expressed genes.

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Laser Microdissection–Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA

Cited by 11 publications

References 67 publications

Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development

Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development

Introduction to Single‐Cell RNA Sequencing

Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti

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