Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs

• Published in: Development genes and evolution Vol. 211; no. 7; pp. 361 - 366
• Main Authors: Imamichi, Y, Lahr, G, Wedlich, D
• Format: Journal Article
• Language: English
• Published: Germany Springer Nature B.V 01-07-2001
• Subjects: Animals; Base Sequence; Cytokeratin; DNA Primers; Ectoderm; Embryo, Nonmammalian - metabolism; Embryogenesis; Endoderm; Evolutionary genetics; Floor plate; Gene expression; Hedgehog protein; Lasers; MyoD protein; Neural cell adhesion molecule; Neural tube; Paraffin; Paraffin Embedding; Polymerase chain reaction; Pressure; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; somites; Xenopus; Xenopus laevis - embryology
• ISSN: 0949-944X; 1432-041X
• DOI: 10.1007/s004270100154

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.