Immunostaining and laser-assisted cell picking for mRNA analysis.
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.
Fink
L
L
Department of Pathology, Justus-Liebig-University, Giessen, Germany. Ludger.Fink@patho.med.uni-giessen.de
Kinfe
T
T
Stein
M M
MM
Ermert
L
L
Hänze
J
J
Kummer
W
W
Seeger
W
W
Bohle
R M
RM
D000469:Alkaline Phosphatase / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007834:Lasers; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D000469:Alkaline Phosphatase