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Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection

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Abstract
Laser capture microdissection (LCM) has become a powerful technique for the isolation of specific cell populations from heterogeneous tissues for gene expression analysis. For standard laser capture microdissection procedures, snap freezing and cryosectioning is the optimal fixation and processing procedure. This sufficiently preserves RNA integrity for downstream molecular analysis. However, in the case of small sized samples, such as early stage embryos, snap freezing and cryosectioning is highly complex and frequently leads to the loss of valuable samples. In the present study, a protocol was optimized to specifically isolate the inner cell mass cells from bovine blastocysts with LCM on chemically fixated paraffin embedded embryos. Bovine blastocysts were fixed for 24h in a methacarn fixative (8 parts methanol, 1 part acetic acid), upon which they were embedded in 2% agarose. After paraffin embedding of the agarose blocks, serial sections of 10 µm were cut and adhered to glass slides. The slides were deparaffinized in xylene, stained with 0.1% cresyl violet in 85% ethanol, and dehydrated in xylene. Subsequently the trophectoderm cells and the inner cell mass cells were separately isolated using LCM. The RNA of the samples was isolated, and subjected to different tests to evaluate the purity of the samples and the quality of the RNA. Expression of Cytokeratin 18 which is only expressed in trophectoderm revealed that the isolated cells were highly pure. An amplicon length assay and a 3’-5’ assay revealed that the RNA quality was sufficient for gene expression analysis with RT-qPCR. In conclusion, LCM on blastocysts is well suited for gene expression analysis of specific cell populations, providing a novel strategy to investigate molecular pathways for pluripotency and cell fate development. This procedure is also compatible with micro-RNA profiling strategies, and the application for downstream transcriptome sequencing is currently being evaluated.
Keywords
gene expression, laser capture microdissection, blastocysts, RT qPCR

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MLA
De Spiegelaere, Ward, et al. “Gene Expression Analysis in Specific Cell Populations of Bovine Blastocysts Using Laser Capture Microdissection.” European Association of Veterinary Anatomists, 29th Congress, Abstracts, 2012.
APA
De Spiegelaere, W., Goossens, K., Stevens, M., Van Soom, A., Peelman, L., Burvenich, C., … Cornillie, P. (2012). Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection. European Association of Veterinary Anatomists, 29th Congress, Abstracts. Presented at the 29th Congress of the European Association of Veterinary Anatomists, Stara Zagora, Bulgaria.
Chicago author-date
De Spiegelaere, Ward, Karen Goossens, Mieke Stevens, Ann Van Soom, Luc Peelman, Christian Burvenich, Bart De Spiegeleer, and Pieter Cornillie. 2012. “Gene Expression Analysis in Specific Cell Populations of Bovine Blastocysts Using Laser Capture Microdissection.” In European Association of Veterinary Anatomists, 29th Congress, Abstracts.
Chicago author-date (all authors)
De Spiegelaere, Ward, Karen Goossens, Mieke Stevens, Ann Van Soom, Luc Peelman, Christian Burvenich, Bart De Spiegeleer, and Pieter Cornillie. 2012. “Gene Expression Analysis in Specific Cell Populations of Bovine Blastocysts Using Laser Capture Microdissection.” In European Association of Veterinary Anatomists, 29th Congress, Abstracts.
Vancouver
1.
De Spiegelaere W, Goossens K, Stevens M, Van Soom A, Peelman L, Burvenich C, et al. Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection. In: European Association of Veterinary Anatomists, 29th Congress, Abstracts. 2012.
IEEE
[1]
W. De Spiegelaere et al., “Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection,” in European Association of Veterinary Anatomists, 29th Congress, Abstracts, Stara Zagora, Bulgaria, 2012.
@inproceedings{3236362,
  abstract     = {{Laser capture microdissection (LCM) has become a powerful technique for the isolation of specific cell populations from heterogeneous tissues for gene expression analysis. For standard laser capture microdissection procedures, snap freezing and cryosectioning is the optimal fixation and processing procedure. This sufficiently preserves RNA integrity for downstream molecular analysis. However, in the case of small sized samples, such as early stage embryos, snap freezing and cryosectioning is highly complex and frequently leads to the loss of valuable samples. In the present study, a protocol was optimized to specifically isolate the inner cell mass cells from bovine blastocysts with LCM on chemically fixated paraffin embedded embryos. Bovine blastocysts were fixed for 24h in a methacarn fixative (8 parts methanol, 1 part acetic acid), upon which they were embedded in 2% agarose. After paraffin embedding of the agarose blocks, serial sections of 10 µm were cut and adhered to glass slides. The slides were deparaffinized in xylene, stained with 0.1% cresyl violet in 85% ethanol, and dehydrated in xylene. Subsequently the trophectoderm cells and the inner cell mass cells were separately isolated using LCM. The RNA of the samples was isolated, and subjected to different tests to evaluate the purity of the samples and the quality of the RNA. Expression of Cytokeratin 18 which is only expressed in trophectoderm revealed that the isolated cells were highly pure. An amplicon length assay and a 3’-5’ assay revealed that the RNA quality was sufficient for gene expression analysis with RT-qPCR. In conclusion, LCM on blastocysts is well suited for gene expression analysis of specific cell populations, providing a novel strategy to investigate molecular pathways for pluripotency and cell fate development. This procedure is also compatible with micro-RNA profiling strategies, and the application for downstream transcriptome sequencing is currently being evaluated.}},
  author       = {{De Spiegelaere, Ward and Goossens, Karen and Stevens, Mieke and Van Soom, Ann and Peelman, Luc and Burvenich, Christian and De Spiegeleer, Bart and Cornillie, Pieter}},
  booktitle    = {{European Association of Veterinary Anatomists, 29th Congress, Abstracts}},
  keywords     = {{gene expression,laser capture microdissection,blastocysts,RT qPCR}},
  language     = {{eng}},
  location     = {{Stara Zagora, Bulgaria}},
  title        = {{Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection}},
  year         = {{2012}},
}