A high-throughput strategy for protein profiling in cell microarrays using automated image analysis.
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.
Strömberg
Sara
S
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Björklund
Marcus Gry
MG
Asplund
Caroline
C
Sköllermo
Anna
A
Persson
Anja
A
Wester
Kenneth
K
Kampf
Caroline
C
Nilsson
Peter
P
Andersson
Ann-Catrin
AC
Uhlen
Mathias
M
Kononen
Juha
J
Ponten
Fredrik
F
Asplund
Anna
A
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
D000906:Antibodies; D011506:Proteins