Dataset Information

A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis.

ABSTRACT:

Background

Gene expression changes that govern essential biological processes can occur at the cell-specific level. To gain insight into such events, laser microdissection is applied to cut out specific cells or tissues from which RNA for gene expression analysis is isolated. However, the preparation of plant tissue sections for laser microdissection and subsequent RNA isolation usually involves fixation and embedding, processes that are often time-consuming and can lower the yield and quality of isolated RNA.

Results

Infection sites of the parasitic plant Cuscuta reflexa growing on its compatible host plant Pelargonium zonale were sectioned using a vibratome and dried on glass slides at 4 °C before laser microdissection. High quality RNA (RQI?>?7) was isolated from 1 mm², 3 mm² and 6 mm² total surface areas of laser microdissection-harvested C. reflexa tissue, with the yield of RNA correlating to the amount of collected material (on average 7 ng total RNA/mm²). The expression levels of two parasite genes previously found to be highly expressed during host plant infection were shown to differ individually between specific regions of the infection site. By drying plant sections under low pressure to reduce the dehydration time, the induced expression of two wound-related genes during preparation was avoided.

Conclusions

Plants can be prepared quickly and easily for laser microdissection by direct sectioning of fresh tissue followed by dehydration on glass slides. We show that RNA isolated from material treated in this manner maintains high quality and enables the investigation of differential gene expression at a high morphological resolution.

SUBMITTER: Olsen S

PROVIDER: S-EPMC6676614 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis.

Olsen Stian S Krause Kirsten K

Plant methods 20190802

<h4>Background</h4>Gene expression changes that govern essential biological processes can occur at the cell-specific level. To gain insight into such events, laser microdissection is applied to cut out specific cells or tissues from which RNA for gene expression analysis is isolated. However, the preparation of plant tissue sections for laser microdissection and subsequent RNA isolation usually involves fixation and embedding, processes that are often time-consuming and can lower the yield and q ...[more]

PMID: 31388345

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Project description:BackgroundSenescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.MethodsWe employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens.ResultsMicroarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified.ConclusionsOur LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.

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Dataset Information

A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis.

Background

Results

Conclusions

Publications

A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis.

Similar Datasets

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