mRNA Expression Analysis of Tissue Sections and Single Cells

Eberwine, James; Kacharmina, Janet Estee; Andrews, Christine L.; Miyashiro, Kevin; McIntosh, Tracy K.; Becker, Kevin G.; Barrett, Tanya; Hinkle, Dave; Dent, Gersham; Marciano, Paolo G.

doi:10.1523/jneurosci.21-21-08310.2001

Cited by 78 publications

(48 citation statements)

References 27 publications

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“…RNA was quantified by using Ribogreen (Molecular Probes). Isolated RNA (5-10 g) was amplified and labeled by using a modified Eberwine protocol (12). Biotin-labeled RNA was hybridized to HG U95A chips, according to the manufacturer's instructions (Affymetrix, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Iourgenko

Zhang

Mickanin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

354

306

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This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.IL-8 ͉ genomics ͉ high-throughput screening ͉ transducer of regulated cAMP response element-binding protein

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Section: Methodsmentioning

confidence: 99%

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Iourgenko

Zhang

Mickanin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

354

306

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“…These procedures essentially entail in vitro RNA transcription using a double-stranded (ds) cDNA template to amplify genes in a linear manner (42,51,52). RNA amplification methods preserve the original quantitative relationship(s) in an amplified gene population, thus facilitating downstream quantitative analysis.…”

Section: Rna Amplification Of Single Cells and Populationsmentioning

confidence: 99%

“…One of the advantages of using single-cell gene expression is that classes of transcripts can be compared across a variety of experimental and disease conditions in discrete cell populations (3,34,52). A paradigm that is quite intriguing is aging, particularly within cell types that are vulnerable to neurodegeneration in late-onset progressive neurodegenerative disorders as well as those implicated in neuropsychiatric disorders (91,92).…”

Section: Single-cell Analysis and Aging: Dopamine Receptor Expressionmentioning

confidence: 99%

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

et al. 2004

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Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by singlecell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively. KEY WORDS:Alzheimer's disease; cDNA microarray; cholinergic basal forebrain; dopamine receptors; expression profiling; protein phosphatases; RNA amplification; schizophrenia; single-cell microaspiration. Abbreviations (note the use of the NCBI-Unigene annotation): 3Rtau, three-repeat tau; 4Rtau, four-repeat tau; ACT, alpha-1-antichymotrypsin; ACTB, beta-actin; AGER, advanced glycosylation end product-specific receptor; APOE, apolipoprotein E; APP, amyloid-beta precursor protein; arc, activity regulated cytoskeletal-associated protein; BAX,

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“…[1][2][3][4][5] An array platform allows investigation of multiple genes (eg, hundreds to thousands) simultaneously. However, fairly large tissue quantities are needed for RNA extraction.…”

mentioning

confidence: 99%

Amplification of RNA transcripts using terminal continuation

Che

Ginsberg

2003

Lab Invest

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A new methodology has been developed to amplify RNA from minute amounts of starting material. Specifically, an efficient means of second-strand (ss) cDNA synthesis using a sequence-specific 'terminal continuation' (TC) method is demonstrated. An RNA synthesis promoter is attached to the 3 0 and/or 5 0 region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications including gene expression profiling, cDNA microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translations and downstream proteomic applications. The TC RNA amplification methodology offers high sensitivity, flexibility, and throughput capabilities. A likely mechanism is that the TC primer binds preferentially to GC-rich CpG islands flanking 5 0 regions of DNA that contain promoter sequences. Following TC RNA amplification, a large proportion of genes can be assessed quantitatively as evidenced by bioanalysis and cDNA microarray analysis in mouse and human postmortem brain tissues.

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mRNA Expression Analysis of Tissue Sections and Single Cells

Cited by 78 publications

References 27 publications

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

Amplification of RNA transcripts using terminal continuation

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