Immunostaining and Laser-Assisted Cell Picking for mRNA Analysis
| Autor: | Ludger Fink, Leander Ermert, Rainer M. Bohle, Jörg Hänze, Werner Seeger, Maria Magdalena Stein, Wolfgang Kummer, Thomas Kinfe |
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| Rok vydání: | 2000 |
| Předmět: |
Cell type
medicine.diagnostic_test Reverse Transcriptase Polymerase Chain Reaction Lasers Cell Antibodies Monoclonal Fluorescent Antibody Technique Cell Separation Cell Biology Biology Alkaline Phosphatase Immunofluorescence Proteinase K Immunohistochemistry Molecular biology Pathology and Forensic Medicine Staining medicine.anatomical_structure Gene expression medicine biology.protein RNA Messenger Molecular Biology Immunostaining Microdissection |
| Zdroj: | Laboratory Investigation. 80:327-333 |
| ISSN: | 0023-6837 |
| Popis: | Isolation of single cells or cell clusters from complex tissue sections has become possible by microdissection techniques. Employing laser-assisted cell picking, cell-specific mRNA analysis of a few isolated cell profiles may be performed. However, microscopic discrimination of different cell types in routinely stained tissue sections is limited, whereas immunostaining enables a more precise access to cells of interest. This approach was noted to interfere with mRNA recovery. To define optimal conditions for mRNA amplification from immunodetected cells, we systematically investigated several potential affectors. Kind of fixation, antibodies and staining reagents, incubation and total processing time, and digestion with proteinase K turned out to influence mRNA stability. We present rapid protocols for immunohistochemistry and immunofluorescence with total incubation times of approximately 25 to 40 minutes and 10 to 20 minutes, respectively, and suggest mRNA amplification without a preceding extraction step. Applying these protocols to oligocellular clusters containing approximately 20 cell profiles and nuclei each from lung and kidney tissue, the highest efficiency rates of mRNA amplification were obtained when combining short-term formalin fixation, reduction of antibody incubation time, application of immunofluorescence, and digestion with proteinase K. Thus, the successful combination of immunostaining and laser-assisted cell picking remarkably improves cell type-specific analysis of gene expression within complex tissues. |
| Databáze: | OpenAIRE |
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