Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 35
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Similarly, when array technologyis used to define an open-ended molecular phenotype, one must be absolutelypositive that the cDNAs present on the array are what they claim to be, and thisgets to the heart of quality control issues at the array manufacturer. One majorproblem with using microarrays to analyze the relative expression level of thousands of cDNAs is that it is impractical to cross check all of or even most of thedata.
If fact, one of the main attractions of using microarrays is the ability to examine expression levels of more cDNAs than possible by any other means.The following account serves to illustrate some important points when considering whether or not to utilize microarray technology and should help to point outpossible problems with the manufacture of arrays. Although our experience isspecific to the ResGen gf300 rat cDNA array marketed by Invitrogen, Inc. the issues and problems we encountered are applicable to any microarray technology.We believe that the wider scientific community may have an interest in knowingabout this particular array manufacturing problem, because these filters are inwide use in studies of gene expression [4–6]. At the very least, our experienceshould serve as a warning to anyone using microarrays that proper experimentsneed to be done to confirm a subset of the array results.Proteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E.
van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-31147 A Cautionary Tale7.2Phase 1: The Original Experimental DesignOur laboratory recently undertook a series of array-based experiments designed toidentify cDNAs that were expressed in different primary cell isolates from the cardiovascular system. Our aim was to profile the complement of cDNAs expressedin cultured rat cardiomyocytes, cardiac fibroblasts, and aortic smooth muscle cells,all isolated from neonatal rats.
We hoped to generate a list of marker genes thatwould make up a molecular phenotype for each of the cardiovascular cell types.Next, we planned to use this information to assay for any “transdifferentiation”events that might take place after overexpression of cardiac-specific transcriptionfactors GATA4, MEF2C, and Nkx2.5 [7–9] in fibroblasts or smooth muscle cells.Furthermore, this approach was expected to identify novel endogenous targets ofthese transcription factors.In order to have the most reliable data possible, we controlled for biological variation by independently preparing RNA from three separate primary cell isolationsof each cell type. For each array hybridization we used fresh arrays purchasedfrom ResGen.
In this way we could control for differences in culture conditionsfrom month to month, and from various lots of printed arrays. We used fresh arrays for every measurement because we had previously determined that ResGenarrays can not be reliably stripped and re-probed.Our first set of array experiments were designed to establish a “baseline” ofgene expression in each to the three cell types. Nine independent RNA isolationswere performed, three from each of three cell types, neonatal cardiomyocytes, cardiac fibroblasts, and aortic smooth muscle cells.We chose to employ microarrays containing 5147 cDNAs spotted onto nylon filters marketed by Invitrogen, Inc.
and manufactured by ResGen, Inc. (Fig. 7.1). Inthe fall of 1999 we purchased a set of filters and the associated probe labeling reagents and hybridization buffers to expedite our studies. These filters were chosenfor several reasons, including the low initial startup cost, the fact that the reagentsand methods for probe preparation and array visualization were already present inour lab, and that at the time, ResGen had by far the largest commercially available collection of rat cDNAs present on an array.
Over the course of a year, weperformed a series of 16 hybridizations to the gf300 arrays and analyzed the output data with custom spreadsheets developed in our lab.Total RNA was extracted from these three primary isolates of neonatal rat cardiovascular cells and used to generate probes for the arrays. Rat gf300 arrays werehybridized to 33P-labeled reverse transcribed probes and all arrays were scannedon a phosphorimager. The images were imported to Pathways 2.0 software package(ResGen, Inc.).
This software was used to identify each of the cDNAs spotted ontothe array and generate a report on the background and signal intensity of eachspot. The raw data reports were exported into Microsoft Excel and processedthrough a series of calculations to subtract background, and normalize each signalto the average signal of the array.
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