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Microarray gene profiling of laser-captured cells: a new tool to study atherosclerosis in mice.

ABSTRACT: Genetically modified mice susceptible to atherosclerosis are widely used in atherosclerosis research. Although the atherosclerotic lesions in these animals show similarities to those in humans, comprehensive expression profile analysis of these lesions is limited by their very small size. In this communication, we have developed an approach to analyze global gene expression in mouse lesions by a combination of (a) laser capture microdissection (LCM) to isolate RNA from specific lesions, and (b) an efficient RNA amplification method that reliably yields sufficient quantities of high quality cRNA for quantitative real-time PCR (qPCR), as well as for microarray analysis. The RNA passed multiple quality controls and the expression profile observed in lesional cells compared with the whole artery encompasses genes that are characteristic of a macrophage/foam cell population. We believe that this method represents a useful new tool for the unbiased analysis of global gene expression of specific sub-regions in atherosclerotic lesions in different rodent models.

SUBMITTER: Paul A

PROVIDER: S-EPMC2613781 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Microarray gene profiling of laser-captured cells: a new tool to study atherosclerosis in mice.

Paul Antoni A Yechoor Vijay V Raja Rajiv R Li Lan L Chan Lawrence L

Atherosclerosis 20080304 2

Genetically modified mice susceptible to atherosclerosis are widely used in atherosclerosis research. Although the atherosclerotic lesions in these animals show similarities to those in humans, comprehensive expression profile analysis of these lesions is limited by their very small size. In this communication, we have developed an approach to analyze global gene expression in mouse lesions by a combination of (a) laser capture microdissection (LCM) to isolate RNA from specific lesions, and (b) ...[more]

PMID: 18289551

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Project description:Alzheimer's disease (AD) is a devastating neurodegenerative disorder that threatens to reach epidemic proportions as our population ages. Although much research has examined molecular pathways associated with AD, relatively few such studies have focused on the disease's critical early stages. In a prior microarray study we correlated gene expression in hippocampus with degree of Alzheimer's disease and found close associations between upregulation of apparent glial transcription factor/epigenetic/tumor suppressor genes and incipient AD. The results suggested a new model in which AD pathology spreads along myelinated axons (Blalock et al., 2004). However, the microarray analyses were performed on RNA extracted from frozen hand-dissected hippocampal CA1 tissue blocks containing both gray and white matter, limiting the confidence with which transcriptional changes in gray matter could be distinguished from those in white matter. Here, we used laser capture microdissection (LCM) to exclude major white matter tracts while selectively collecting CA1 hippocampal gray matter from formalin-fixed, paraffin-embedded (FFPE) hippocampal sections of the same subjects assessed in our prior study. Microarray analyses of this gray matter-enriched tissue revealed many transcriptional changes similar to those seen in our past study and in studies by others, particularly for downregulated neuron-related genes. Additionally, the present analyses identified several previously undetected pathway alterations, including downregulation of molecules that stabilize ryanodine receptor Ca2+ release and upregulation of vasculature development. Conversely, we found a striking paucity of the upregulated changes in the putative glial and growth-related genes that had been strongly overrepresented in the prior mixed-tissue study. We conclude that FFPE tissue can be a reliable resource for microarray studies of brain tissue, that upregulation of growth-related epigenetic/transcription factors during incipient AD is predominantly localized in and around white matter (supporting our prior findings and model), and that novel alterations in vascular and ryanodine receptor-related pathways in gray matter are closely associated with incipient AD.

Project description:BACKGROUND:Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. RESULTS:In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. CONCLUSIONS:Our study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

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Microarray gene profiling of laser-captured cells: a new tool to study atherosclerosis in mice.

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Microarray gene profiling of laser-captured cells: a new tool to study atherosclerosis in mice.

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