title: A high-throughput strategy for protein profiling in cell microarrays using automated image analysis.
abstract: 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.
authors.affiliation: Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Strömberg
authors.forename: Marcus Gry
authors.initials: MG
authors.lastname: Björklund
authors.forename: Caroline
authors.initials: C
authors.lastname: Asplund
authors.forename: Anna
authors.initials: A
authors.lastname: Sköllermo
authors.forename: Anja
authors.initials: A
authors.lastname: Persson
authors.forename: Kenneth
authors.initials: K
authors.lastname: Wester
authors.forename: Caroline
authors.initials: C
authors.lastname: Kampf
authors.forename: Peter
authors.initials: P
authors.lastname: Nilsson
authors.forename: Ann-Catrin
authors.initials: AC
authors.lastname: Andersson
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlen
authors.forename: Juha
authors.initials: J
authors.lastname: Kononen
authors.forename: Fredrik
authors.initials: F
authors.lastname: Ponten
authors.forename: Anna
authors.initials: A
authors.lastname: Asplund
mesh_terms: D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012984:Software
chemical_list: D000906:Antibodies; D011506:Proteins