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 ce ll 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 ena bles a more precise access to cells of interest. This approach was noted to interfere with mRNA recovery. To define optimal conditions for mRNA amplif ication from immunodetected cells, we systematically investigated several p otential affectors. Kind of fixation, antibodies and staining reagents, inc ubation and total processing time, and digestion with proteinase K turned o ut to influence mRNA stability. We present rapid protocols for immunohistoc hemistry and immunofluorescence with total incubation times of approximatel y 25 to 40 minutes and 10 to 20 minutes, respectively, and suggest mRNA amp lification without a preceding extraction step. Applying these protocols to oligocellular clusters containing approximately 20 cell profiles and nucle i each from lung and kidney tissue, the highest efficiency rates of mRNA am plification were obtained when combining short-term formalin fixation, redu ction of antibody incubation time, application of immunofluorescence, and d igestion with proteinase K. Thus, the successful combination of immunostain ing and laser-assisted cell picking remarkably improves cell type-specific analysis of gene expression within complex tissues.