Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level.
Morphological study and identification of cell differentiation within tissues are the basis for assessment of physiological and pathological processes. Applying molecular techniques, the analysis of tissue homogenates may lead to masking of genetic deviations or expression changes of an individual cell type by the bulk of surrounding cells. To overcome the tissue heterogeneity, cells have to be isolated selectively. Therefore, microdissection techniques were developed making retrieval of target cells simple, rapid and precise. Presenting an overview of our approaches, it is demonstrated that single cell isolation is often preconditional for the investigation of splicing isoform expression. To reveal gene regulation combining microdissection of few cells and real-time RT-PCR allows to determine relative mRNA expression in a cell type-specific manner, even after immunofluorescence staining of target cells. Combination with RNA preamplification techniques and micro arrays results in cell type- or compartment-specific expression profiles that especially differ from those of tissue homogenates when minor represented cell types are investigated. For proteomic biomarker screening, the application of mass spectrometry techniques (SELDI/MALDI TOF) turned out to be feasible in combination with microdissected minute amounts of tissues. However, further identification of marker peaks still needs a remarkable effort. Strategies to deal with this problem are presented. In consequence, the isolation of cells or cell types allows a more accurate investigation of complex tissues and gives deeper insight to regulation processes and crosstalk of the respective cells.
Fink
Ludger
L
Department of Pathology, University of Giessen Lung Center, Langhansstr 10, 35392 Giessen, Germany. ludger.fink@patho.med.uni-giessen.de
Kwapiszewska
Grazyna
G
Wilhelm
Jochen
J
Bohle
Rainer M
RM
D000818:Animals; D005786:Gene Expression Regulation / Q000502:physiology*; D023281:Genomics; D007834:Lasers; D008168:Lung / Q000166:cytology*; D042282:Microdissection / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011506:Proteins; D012333:RNA, Messenger