RESUMO
A histoanatomical context is imperative in an analysis of gene expression in a cell in a tissue to elucidate physiological function of the cell. In this study, we made technical advances in fluorescence laser microdissection (LMD) in combination with the absolute quantification of small amounts of mRNAs from a region of interest (ROI) in fluorescence-labeled tissue sections. We demonstrate that our fluorescence LMD-RTqPCR method has three orders of dynamic range, with the lower limit of ROI-size corresponding to a single cell. The absolute quantification of the expression levels of the immediate early genes in an ROI equivalent to a few hundred neurons in the hippocampus revealed that mice transferred from their home cage to a novel environment have distinct activation profiles in the hippocampal regions (CA1, CA3, and DG) and that the gene expression pattern in CA1, but not in the other regions, follows a power law distribution.
Assuntos
Perfilação da Expressão Gênica/métodos , Hipocampo/metabolismo , Lasers , Microdissecção/métodos , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/embriologia , Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/embriologia , Região CA3 Hipocampal/metabolismo , Giro Denteado/citologia , Giro Denteado/embriologia , Giro Denteado/metabolismo , Feminino , Fluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Hipocampo/citologia , Hipocampo/embriologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Proteína Vermelha FluorescenteRESUMO
In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.
