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IFN-Gamma keratinocytes

ABSTRACT: An experiment comparing control versus IFN-g treated skin cells (one design factor on two levels). To evaluate the biological and the experimental variability, the following experimental design was planned as shown in Figure 1: "Control" and "IFN-g stimulated" keratinocytes were cultured in triplicate using three individual petri Æ10 culture dishes and RNA was independently extracted from each cultured replicate. For each RNA sample, cRNA synthesis was performed in triplicate and each cRNA pool was hybridized to an Affymetrix U133 Gene Chip. Keywords: ordered

ORGANISM(S): Homo sapiens

PROVIDER: GSE1132 | GEO | 2004/09/01

SECONDARY ACCESSION(S): PRJNA87247

REPOSITORIES: GEO

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Project description:The data in this series forms the basis for the analysis presented in the above named paper. In this study, we used cDNA microarrays to ask if changes in gene expression are observed in response to a dietary shift in Drosophila melanogaster, a dietary generalist. Methods: Flies used in this study were derived from a multifemale collection at Iraklion, Crete, Greece, in August 2002 and reared in the laboratory for several generations. Three replicates were performed. At the start of each replicate, we collected 300 newly eclosed adults, separated them by sex, and stored them at low density in shell vials with standard cornmeal media for 3 days. Sexually mature males and females were placed together and allowed to mate. Mated females were allowed to oviposit overnight in chambers containing cornmeal medium filled Petri dishes (http://flyfood.arl.arizona.edu/cornmeal.php3). Hatching first instar larvae were reared for 36 hours on cornmeal medium in Petri dishes and then transferred to one of two conditions: 1) a control Petri dish consisting of cornmeal medium, or 2) an experimental Petri dish containing pure mashed banana. After 24 hours, larvae were plucked from these dishes, washed, transferred to microcentrifuge tubes in groups of 20, and flash frozen in liquid nitrogen. All samples were stored at -80C until RNA extraction. RNA was extracted from each tube using a Qiagen RNeasy kit (Qiagen, Inc., standard protocol). We used cDNA microarrays printed by the Genetic Analysis and Technology Core Custom Microarray Facility at the University of Arizona to assess expression levels. Printing material was amplified from BDGP Unigene library v1.0 containing 6K ESTs. Products were purified using Millipore Multiscreen filtration plates and re-suspended in 50%DMSO solution. Each product was printed in triplicate on Corning GAPSII aminosilane slides using a Virtek Chipwriter PRO microarray spotter. Between 10-15 micrograms of RNA were used for each reaction. RNA was reverse transcribed and labeled with one of two fluorescent dyes, Cy3 or Cy5 (details of protocol at http://gatc.arl.arizona.edu/Services/Microarray/aminoallyllabelling.html). Hybridization was completed on a Genomic Solutions Gentac automated hybridization station. Labeled material was allowed to hybridize to the array for 16 hours at 60°C. Wash conditions were: 1) low stringency wash twice at 50°C for 40seconds (1X SSC/ 0.2% SDS), 2) medium stringency wash twice at 42°C for 40 seconds ( 0.1X SSC/ 0.2% SDS), 3) high stringency wash twice at 42°C for 40 seconds (0.1X SSC). Hybridized arrays were scanned using the Applied Precision ArrayworxE slide scanner. We performed three independent hybridizations and three dye flips for a total of six arrays (2 treatments x 2 dyes x 3 replications = 12 samples, for 6 arrays). Each replicate consisted of an independent biological sample isolated on separate days. Identification of series: There are 18 series in this experiment. The second number in each series record represents the slide number (there were six slides used in this experiment). The third number in each series record represents the set on a given slide. For instance, for series record 331_2_1, these data correspond to slide 2, set 1. Each slide has 3 series numbers assoicated with it, representing set 1,2, or 3 on the slide. Each slide was paired with another in a "dye flip" experiment; thus, slides 2 and 3 were paired, slides 25 and 28 were paired, and slides 29 and 30 were paired. Keywords = dietary shifts Keywords = ecological genomics Keywords: repeat sample

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IFN-Gamma keratinocytes

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