Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs.
One of the key end points for understanding the molecular basis of embryogenesis is the analysis of spatiotemporal patterns of gene expression. Methodical limitations due to low mRNA levels often prevent a tissue-specific resolution. In this study, we developed an improved laser microdissection technique and RT-PCR that allows marker gene detection in small tissue areas from sections of formalin-fixed paraffin-embedded Xenopus embryos. Tissue pieces were isolated by laser microbeam microdissection and captured by laser pressure catapulting. Neither laser treatment nor conventional histological or immunochemical staining impaired subsequent RNA analysis. Transcripts of tissue-specific marker genes such as endodermin (endoderm), epidermal cytokeratin (epidermal ectoderm), N-CAM (neural tube), myoD (somites), and sonic hedgehog (floor plate) were amplified by nested RT-PCR analysis from small areas of single sections.
Imamichi
Y
Y
Department of Biochemistry, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Lahr
G
G
Wedlich
D
D
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D007834:Lasers; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis / Q000196:embryology
D017931:DNA Primers; D012333:RNA, Messenger