Laser capture microdissection (LCM) for analysis of gene expression in specific tissues during embryonic epithelial-mesenchymal transformation.
The analysis of gene expression in developing organs is a valuable tool for the assessment of genetic fingerprints during the various stages of tissue differentiation and epithelial-mesenchymal transformation (EMT). However, the variety of differentiating cells and the close association of epithelial and mesenchymal cells makes it difficult to extract protein and mRNA from specific cells and tissue and, thus, to assign expressed genes to specific cell populations. We report here the analysis of LEF1 mRNA in epithelial and mesenchymal cells isolated by LCM from different stages of EMT during development of the mouse palate and describe our techniques in detail. By applying a laser capture microdissection (LCM) technique and real-time polymerase chain reaction, we were able to determine mRNA levels that accurately reflect changes in gene expression in specific cells. The sensitivity of the technique is remarkable. Indeed, the mRNAs can be detected for many proteins too low in abundance to stain with antibodies. These techniques will enable embryologists to collect homogeneous groups of cells from heterogeneous populations in developing organs, which otherwise would not be available for gene analysis.
Nawshad
A
A
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
LaGamba
D
D
Olsen
B R
BR
Hay
E D
ED
D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry; D004622:Embryo, Mammalian; D004848:Epithelium / Q000196:embryology / Q000378:metabolism*; D015870:Gene Expression; D007834:Lasers; D051821:Lymphoid Enhancer-Binding Factor 1; D008648:Mesoderm / Q000378:metabolism*; D051379:Mice; D042282:Microdissection / Q000379:methods*; D009928:Organ Specificity; D010159:Palate / Q000196:embryology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000737:chemistry
D004268:DNA-Binding Proteins; C497574:Lef1 protein, mouse; D051821:Lymphoid Enhancer-Binding Factor 1; D012333:RNA, Messenger; D014157:Transcription Factors