Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 4
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van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-331Large Scale Expression Profilingin Cardiovascular Disease Using Microarrays:Prospects and PitfallsMartina Schinke, Lauren Riggi, Atul J. Butte, and Seigo Izumo1.1DNA Microarray TechnologiesMicroarray technologies have revolutionized the way we identify gene expressionchanges in biological events [1] and complex diseases [2]. Instead of looking at ahandful of genes, recent technology advances allow us to scan up to 12,000 transcripts at once. As the draft sequencing of the human genome has been completed and it becomes clear that it merely contains 33,000 genes [3], it is hopedthat global expression profiling integrated with other types of genomic information will fully describe the regulation of gene-expression networks and how theymalfunction in disease.In this chapter we will provide an overview of current microarray technologies,describe how to design meaningful microarray experiments, and address the typeof answers it can be expected to provide.
We also consider the pitfalls and shortcomings of the technology, and different approaches of data analysis developed todeal with the data avalanche. Finally, we review current literature to see how microarray studies have helped us to decipher the transcriptional regulatory networkunderlying cardiovascular diseases.1.1.1cDNA Microarrays or Oligonucleotide Arrays?The construction of gene expression databases requires technologies that can accurately and reproducibly measure changes in global mRNA expression levels.Ideally, these technologies should be able to screen all gene transcripts, be applicable across a wide range of cell and tissue types, require minimal amounts of biological material, and be capable of processing large number of samples.
Althoughseveral different technology platforms have been developed [4–9], each comes withits own set of advantages and limitations in meeting these stringent requirements(Fig. 1.2). The two most commonly used platforms are complementary DNA(cDNA) microarrays and high-density oligonucleotide arrays.Proteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E. van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co.
KGaA, WeinheimISBN: 3-527-30596-341 Microarray Expression Profiling in Cardiovascular Disease1.1 DNA Microarray TechnologiescDNA microarrays have been developed by several academic groups as well ascommercial suppliers. In general, cDNA libraries or clone collections are amplified by polymerase chain reaction (PCR) at an average product size of 1,000 nucleotides in length. These PCR products are printed in a two-dimensional gridonto glass slides or nylon membranes as spots at defined locations. Spots are typically 100–300 lm in size and are spaced about the same distance apart.
Usingthis technique, arrays consisting of more than 30,000 cDNAs can be fitted ontothe surface of a conventional microscope slide (Fig. 1.2).The process of spotting is generally not accurate enough to allow direct comparison between different arrays. Therefore, the two RNA samples from tissues to becompared are typically used to generate first-strand cDNA targets labeled with twodifferent fluorescent dyes, for example Cy3 and Cy5.
These are then purified,pooled and hybridized to the same array, resulting in competitive binding of thedifferentially labeled cDNAs to the arrayed sequences (Fig. 1.1). After hybridization and washing, the slide is scanned in a high-resolution confocal fluorescentscanner using two different wavelengths corresponding to the dyes used, and theintensity of the same spot in both channels is compared. From these measurements, a ratio of transcript levels for each gene represented on the array can becalculated. In order to be able to compare results from different arrays to eachother, a reference RNA, e.g.
a mixture of all the samples of one experiment or acommercially available standard, is used to normalize the Cy3 and Cy5 intensities(see 2.2 Comparing expression data).Protocols for total RNA isolation and nucleic acid labeling are available on theinternet from a variety of academic laboratories (Tab. 1.1). To achieve the most linear relationship between starting material and labeled probe, incorporation offluorescent-labeled nucleotides during first strand cDNA synthesis has been themethod of choice. However, this requires up to 100 lg of total RNA as startingmaterial, which excludes studies using primary cells or human tissues of limitedavailability.3Fig. 1.1 Schematic overview of cDNA andoligonucleotide microarray sample preparation.
Total RNA is isolated from tissues of theexperimental sample (e.g. hypertrophic heart),and from the control sample (e.g. normalheart). A For cDNA microarrays, RNA fromboth samples is reverse transcribed into single-stranded cDNA in the presence of twofluorescent dyes (such as Cy3 and Cy5). Bothsamples are mixed in the hybridization bufferand hybridized to the array, usually under acoverslip.
After washing, array slides arescanned by a high-resolution confocal fluorescence scanner with two wavelengths corresponding to the two fluorescent dyes used,and independent images for the control andexperimental channels are generated. Signalintensities for both dyes are used to calculatethe ratios of mRNA abundance. B Total RNAis isolated from the experimental and controltissue sample, and cDNA is synthesizedusing an Oligo-dT primer that carries the T7promotor sequence. T7 DNA polymerase isthen used for in vitro transcription with biotin-labeled nucleotides that become incorporated into the cRNA.
Each labeled cRNA isthen hybridized to an array in a rotating hybridization oven. After hybridization, the array iswashed and stained using a fluorescent dyecoupled to streptavidin. Arrays are scannedand signal intensities are converted into Average Difference (AvgDiff) values or Signals.Modified from [64].561 Microarray Expression Profiling in Cardiovascular DiseaseTab. 1.1 Links to Microarray Resources. This table provides a selection of commercial and aca-demic facilities and resources related to microarray technologiesMicroarray Core Facilitieshttp://sequence-www.stanford.edu/Stanford DNA Sequencing and Technology Centerhttp://www.cgr.harvard.eduBauer Center for Genomics Researchhttp://www.nhgri.nih.gov/DIR/Microarray/main.html National Human Genome Research Institutehttp://microarrays.comMicroarrays Inc., Nashville, TNPublic Microarray Databaseshttp://pga.lbl.gov/PGA/PGA_inventory.htmlhttp://www.cardiogenomics.orghttp://www.dnachip.orghttp://www.ncbi.nlm.nih.gov/geo/http://www.ebi.ac.uk/microarray/ArrayExpress/Protocols and Softwarehttps://www.affymetrix.com/http://biosun1.harvard.edu/complab/dchip/http://cmgm.stanford.edu/pbrown/protocols/index.htmlhttp://www.microarrays.orghttp://www.tigr.org/software/http://rana.lbl.gov/EisenSoftware.htmhttp://www.biodiscovery.com/http://ep.ebi.ac.uk/NHLBI Programs for Genomic Applications Inventory SiteRaw data and normalized data related tocardiomyopathies in mouse models andhumans using Affymetrix GeneChipsTMMicroarray data storage, image files,data retrieval, analysis, and visualizationGene expression and hybridization arraydata repository, and online resource forthe retrieval of gene expression datafrom any organism or artificial sourcePublic database for microarray basedgene expression dataAffymetrix online resource center forGeneChip arraysdCHIP, freely available analysis tool forAffymetrix GeneChip arraysA guide to cDNA microarraysPublic source for microarray protocolsand softwareSoftware tools freely available to the scientific communitySoftware tools for image analysis, andcluster analysis and visualizationCommercial software products for microarray research and analysisSet of tools for clustering, analysis andvisualization of gene expression dataDevelopment of standards for array experiment annotation and data representationhttp://www.mged.orgMicroarray Gene Expression DatabaseGroup1.1 DNA Microarray TechnologiesSchematic overview of the manufacturing process, and a summary of advantagesand disadvantages of cDNA versus oligonucleotide arrays.
A Inserts from cDNA librariesor clone collections are amplified by PCR.PCR products are spotted or printed by anarrayer or through ink-jet technology ontoglass slides or nylon membranes at specifiedintervals, for example by using chemical linkers that covalently bind the DNA to theglass slide. B On Affymetrix GeneChipsTM,each gene is represented by a probe set of16–20 short oligonucleotides (25mers) thatFig.
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