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A high-throughput strategy for protein profiling in cell microarrays using automated image analysis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2007 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 13, 2142-2150 p.Article in journal (Refereed) Published
Abstract [en]

Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. In a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.

Place, publisher, year, edition, pages
2007. Vol. 7, no 13, 2142-2150 p.
Keyword [en]
Antibody proteomics, Automated image analysis, Cell line, Immunohistochemistry, Tissue microarray
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-97143DOI: 10.1002/pmic.200700199ISI: 000248013700002PubMedID: 17549799OAI: oai:DiVA.org:uu-97143DiVA: diva2:171951
Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2011-01-28Bibliographically approved
In thesis
1. Antibody-based Profiling of Expression Patterns using Cell and Tissue Microarrays
Open this publication in new window or tab >>Antibody-based Profiling of Expression Patterns using Cell and Tissue Microarrays
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.

To analyze protein expression in in vitro cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells in vivo. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types.

Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.

In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 51 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 344
Pathology, antibody-based proteomics, tissue microarray, cell line, immunohistochemistry, melanocytes, image analysis, biomarker, Patologi
urn:nbn:se:uu:diva-8680 (URN)978-91-554-7183-5 (ISBN)
Public defence
2008-05-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 10:00
Available from: 2008-04-25 Created: 2008-04-25Bibliographically approved

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