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Gene expression in normal tissues to validate the first generation porcine oligonucleotide microarray

ABSTRACT: Lung, liver, muscle (Semitendinosus), small intestine (duodenum; approximately 5 cm from stomach) were collected from six cross-bred white pigs (three 15 week old and three 17 week old). Total RNAs were isolated from these tissues to study gene expression by using the first generation of porcine long oligonucleotide microarray. In the experimental design, tissue samples from the six animals were assigned to six loops (three for younger animals and three for older animals). One sample from each of the four tissues from a single animal was used in each loop. The order of the tissues in each loop was varied so that all pairs of tissues were represented together on a slide an equal number of times. Dye balance was used throughout so that each tissue was measured an equal number of times with each dye. Data from 48 measurements from 24 slides were collected, with 12 measurements for each tissue. The mixed model included fixed effects for dye, tissue, animal age, and age-by-tissue interaction as well as random effects for slide, animal, and animal-by-tissue interaction. Genes with expression significantly higher in one tissue than in each of the others will be referred to as tissue-selective genes. To identify such genes, pair-wise comparisons of normalized signal intensities between tissues were conducted as part of our mixed model analyses for each gene. Keywords: repeat sample

ORGANISM(S): Sus scrofa

PROVIDER: GSE2335 | GEO | 2005/03/05

SECONDARY ACCESSION(S): PRJNA91557

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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Gene expression in normal tissues to validate the first generation porcine oligonucleotide microarray

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