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Immunostaining
and Laser-Assisted Cell Picking for mRNA Analysis |
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Ludger Fink, Thomas Kinfe, Maria Magdalena Stein, Leander Ermert, Jörg
Hänze, Wolfgang Kummer, Werner Seeger, and Rainer Maria Bohle
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Departments
of Pathology (LF, TK, MMS, LE, RMB) and Internal Medicine (LF, JH, WS),
and the Institute for Anatomy and Cell Biology (WK), Justus-Liebig-University,
Giessen, Germany |
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SUMMARY: 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. (Lab Invest 2000, 80: 327-333)
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