Gene expression analysis of a single or few cells.
The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi-quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.
Klein
Christoph A
CA
Ludwig-Maximilians-University of Munich, Munich, Germany.
Zohlnhöfer
Dietlind
D
Petat-Dutter
Karina
K
Wendler
Nicole
N
D000818:Animals; D001665:Binding Sites; D001706:Biopsy; D002138:Calibration; D002477:Cells / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D007202:Indicators and Reagents; D042282:Microdissection; D009838:Oligodeoxyribonucleotides; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT)