Identification of differentially expressed genes in mouse development using differential display and in situ hybridization.
A common problem in developmental biology is the isolation of genes that are expressed differentially between two closely matched tissue populations, for example, between wild-type and mutant mouse embryos generated by targeted gene disruption. Typically, the applicable experimental methodologies are limited by the amount of mRNA that can be obtained for cDNA library construction and/or expression analysis. Differential display is a polymerase chain reaction (PCR)-based RNA fingerprinting technique that is ideally suited for the identification of differentially expressed transcripts when only limiting amounts of tissue are available, as is frequently encountered in studies of vertebrate development. Here, I describe protocols for differential display using arbitrarily primed reverse transcription PCR and for the subsequent verification of differential gene expression that are adapted for molecular genetic studies of mouse embryogenesis. The overall strategy involves two steps: First, RNA samples isolated from two nearly identical populations of embryos or microdissected embryonic tissues are compared by differential display, and candidate differentially expressed transcripts are identified. Second, these candidate transcripts are analyzed for differential expression in vivo using nonradioactive whole-mount or section in situ hybridization. In principle, this strategy permits the efficient isolation of genes that are differentially expressed during early mouse embryogenesis.
Shen
M M
MM
Center for Advanced Biotechnology and Medicine and Department of Pediatrics, University of Medicine and Dentistry of New Jersey--Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, New Jersey 08854, USA. mshen@camb.rutgers.edu
D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D018076:DNA, Complementary