Gene Expression Profiling of Primary Tumor Cell Populations Using Laser Capture Microdissection, RNA Transcript Amplification, and GeneChip® Microarrays

Luzzi, Veronica; Holtschlag, Victoria; Watson, Mark A.

doi:10.1385/1-59259-853-6:187

Cited by 10 publications

(7 citation statements)

References 15 publications

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“…2A, B, respectively). The 28S∶18S rRNA ratio in the samples obtained was around 2∶1, which is excellent for RNA isolated from tissues by LMD [13]. Furthermore, the yield of RNA from the fixed tissue (6–9 ng from 3,000–5,000 cells) exceeded that from frozen tissue (4–7 ng from 3,000–5,000 cells), probably due to inactivation of RNases by aldehyde fixation.…”

Section: Resultsmentioning

confidence: 77%

Gene Expression Analysis of In Vivo Fluorescent Cells

et al. 2007

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BackgroundThe analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.Methodology/Principal FindingsWe developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).Conclusions/SignificanceOur technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.

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Section: Resultsmentioning

confidence: 77%

Gene Expression Analysis of In Vivo Fluorescent Cells

et al. 2007

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“…Linear amplification of transcript and hybridization to high-density oligonucleotide microarrays (Affymetrix Mouse Genome U74A) were performed as described previously (19). Microarray normalization and statistical analysis were performed using packages from the Bioconductor project executed in the R programming environment (20).…”

Section: Rna Analysismentioning

confidence: 99%

Airway Epithelial versus Immune Cell Stat1 Function for Innate Defense against Respiratory Viral Infection

Shornick

Wells

Zhang

et al. 2008

The Journal of Immunology

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The epithelial surface is often proposed to actively participate in host defense, but the evidence remains circumstantial. Here we use a mouse paramyxovirus (Sendai virus; SeV) to show that a prominent early event in respiratory infection is activation of Stat1 in airway epithelial cells. Stat1−/– mice developed illness that resembled severe paramyxoviral respiratory infection in humans and was characterized by increased viral replication and neutrophilic inflammation in concert with overproduction of TNF‐α and MIP‐2. Poor control of viral replication as well as TNF‐α and MIP‐2 overproduction were mimicked by infection of Stat1−/– airway epithelial cells in vitro. TNF‐α drives the MIP‐2 response, since it can be reversed by TNF‐α blockade both in vitro and in vivo. These findings pointed to an epithelial defect in Stat1−/– mice. Indeed, Stat1−/– mice reconstituted with wild‐type bone marrow were still susceptible to infection with SeV whereas wild‐type mice that received Stat1−/– bone marrow retained resistance to infection. The susceptible epithelial Stat1−/– chimeric mice also exhibited increased viral replication as well as excessive neutrophils, MIP‐2, and TNF‐α in the airspace. These findings provide some of the most definitive evidence for the role of barrier epithelial cells in innate immunity to common pathogens, particularly in controlling viral replication. Funding: NIH & Parker B. Francis Foundation

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“…Within the context of drug discovery, transcriptional profiling is commonly performed on whole tissue or smaller defined regions of tissue, such as purified cells that are unique to the particular tissue of interest [11,12], small morphologically distinct regions isolated via physical microdissection [13,14] or laser capture microdissection [15]. The following paragraphs will describe some of the uses of data generated by the transcriptional profiling of whole and fractionated tissues using examples from the literature.…”

Section: Tissue Expression Profilingmentioning

confidence: 99%

Gene Expression Profiling and its Practice in Drug Development

Chengalvala

Chennathukuzhi²,

Johnston³

et al. 2007

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The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.

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Gene Expression Profiling of Primary Tumor Cell Populations Using Laser Capture Microdissection, RNA Transcript Amplification, and GeneChip® Microarrays

Cited by 10 publications

References 15 publications

Gene Expression Analysis of In Vivo Fluorescent Cells

Gene Expression Analysis of In Vivo Fluorescent Cells

Airway Epithelial versus Immune Cell Stat1 Function for Innate Defense against Respiratory Viral Infection

Gene Expression Profiling and its Practice in Drug Development

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