Single‐cell dissection and microdroplet chemistry

Outlaw, William H.; Zhang, Shuqiu

doi:10.1093/jexbot/52.356.605

Cited by 36 publications

(23 citation statements)

References 71 publications

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“…Several general approaches have been employed to focus this to cellular and even sub-cellular resolution (Kehr 2001). These include in situ hybridization (McFadden 1994), elegant hand-dissection (Outlaw and Zhang 2001), and advanced laser-capture micro-dissection (Simone et al 1998). Promoter-reporter expression systems (for reviews, see Guivarch et al 1996;Hanson and Ko¨hler 2001), although popular, have certain drawbacks.…”

Section: Introductionmentioning

confidence: 99%

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

Laval

Koroleva

Murphy

et al. 2002

Planta

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The contents of single plant cells can be sampled using glass microcapillaries. By combining such single-cell sampling with reverse transcription-polymerase chain reaction (RT-PCR), transcripts of individual genes can be identified and, in principle, quantified. This provides a valuable technique for the analysis and quantification of the intercellular distribution of gene expression in complex tissues. In a proof-of-principle study, the cellular locations of the transcripts of the eight isoforms of actin (ACT) expressed in Arabidopsis thaliana (L.) Heynh. were analyzed. Cell sap was extracted from epidermal and mesophyll cells of leaves of 3-to 4-week-old plants. Single-cell (SC)-RT-PCR was used to amplify the actin transcripts using specific primer pairs for ACT1, 2, 3, 4, 7, 8, 11 and 12. Only ACT2 and ACT8 were found in epidermal and in mesophyll cells. In individual trichomes, in addition to ACT2 and ACT8, ACT7 and ACT11 transcripts were detectable. By employing the already well-characterized actin system we demonstrate the practicality and power of SC-RT-PCR as a technique for analyzing gene expression at the ultimate level of resolution, the single cell.

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Section: Introductionmentioning

confidence: 99%

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

Laval

Koroleva

Murphy

et al. 2002

Planta

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“…The latter approach is usually limited to use in Arabidopsis and rice and maize plants, which are amenable to transformation and are easily handled in large quantities (Evrard et al., ; Li & Arumuganathan, ; Li, Arumuganathan, Gill, & Song, ). Some techniques applied to animal tissue, including sectioning of frozen tissue blocks to select for specific cell populations, irradiation of stained sections to destroy unwanted cells, and microdissection with manual tools such as sharp needles (Emmert‐Buck et al., ; Outlaw & Zhang, ; Schrader et al., ), are not applicable to plant tissue. Further, plant tissue and cells present specific challenges in comparison to animal tissue due to the presence of plant cell walls, vacuoles, chloroplasts, and secondary metabolites, making LCM a better choice (Cosgrove, ; Giannella et al.…”

Section: Commentarymentioning

confidence: 99%

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

et al. 2018

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In order to study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique does not require separation of cells by enzymatic digestion of any kind, does not require cell-specific reporter genes, and allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capture of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2018 by John Wiley & Sons, Inc.

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“…, ; Jones et al . ; Outlaw & Lowry ; Shen & Outlaw ; Outlaw & Zhang ). Despite this, comparisons of the photosynthetic cell types (mesophyll and guard cells) with their non‐photosynthetic counterparts (e.g.…”

Section: Introductionmentioning

confidence: 99%

Contributions of photosynthetic and non‐photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature

Long

Bahar

Atkin

2015

Plant Cell & Environment

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In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.

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Single‐cell dissection and microdroplet chemistry

Cited by 36 publications

References 71 publications

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

Contributions of photosynthetic and non‐photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature

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