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Sample GSM183163 Query DataSets for GSM183163
Status Public on Apr 20, 2007
Title Reovirus MyRV2-CpC18 infection (Sample B)
Sample type RNA
 
Channel 1
Source name Reovirus MyRV2-CpC18 infected strain EP155
Organism Cryphonectria parasitica
Characteristics Rovirus MyRV2-CpC18 infected strain EP155 grown on PDA with cellophane overlay (sample B).
Biomaterial provider Hillman lab
Treatment protocol Rovirus MyRV2-CpC18 infection
Growth protocol Grow at 22-24 C with a 12-h light-dark cycle at 1300-1600 lx.
Extracted molecule total RNA
Extraction protocol Cultures used for RNA isolation were grown on PDA-cellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen and then immediately grinding the mycelia into a fine powder by using a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol) at a ratio of 4 ml of buffer per g of mycelia. This step was followed sequentially by extraction with equal volumes of water-saturated phenol, acid phenol-chloroform-isoamyl alcohol (25:24:1) (pH 4.5), and chloroform. Single-stranded RNA was precipitated on ice for 2 h by the addition of LiCl to a final concentration of 2 M. The single-stranded RNA precipitate was collected by centrifugation, resuspended in 2 ml of double-distilled H2O, reprecipitated by the addition of 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), and incubated for 30 min at -20°C. The RNA was collected by centrifugation, washed with 2 ml of ice-cold 75% ethanol, dried, and treated with 2 U of RQ1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37°C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl of double-distilled H2O.
Label Cy3
Label protocol Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy3-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.
 
Channel 2
Source name Wild type strain EP155
Organism Cryphonectria parasitica
Characteristics Wild type strain EP155 grown on PDA with cellophane overlay (sample B).
Biomaterial provider Hillman lab
Treatment protocol No treatment
Growth protocol Grow at 22-24 C with a 12-h light-dark cycle at 1300-1600 lx.
Extracted molecule total RNA
Extraction protocol Cultures used for RNA isolation were grown on PDA-cellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen and then immediately grinding the mycelia into a fine powder by using a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol) at a ratio of 4 ml of buffer per g of mycelia. This step was followed sequentially by extraction with equal volumes of water-saturated phenol, acid phenol-chloroform-isoamyl alcohol (25:24:1) (pH 4.5), and chloroform. Single-stranded RNA was precipitated on ice for 2 h by the addition of LiCl to a final concentration of 2 M. The single-stranded RNA precipitate was collected by centrifugation, resuspended in 2 ml of double-distilled H2O, reprecipitated by the addition of 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), and incubated for 30 min at -20°C. The RNA was collected by centrifugation, washed with 2 ml of ice-cold 75% ethanol, dried, and treated with 2 U of RQ1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37°C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl of double-distilled H2O.
Label Cy5
Label protocol Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy5-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.
 
 
Hybridization protocol Printed slides were prepared for hybridization by the addition of 30 μl of prehybridization solution, which contained 50% formamide, 6X SSPE (1X SSPE is 0.15 M NaCl, 0.001 M NaH2PO4 and 0.001 M EDTA), 0.5% SDS, 5X Denhardt’s solution, and 100 μg of salmon sperm DNA/ml, to the arrayed surface of a glass slike (covered with a coverslip to evenly distribute the prehybridization solution). The slide was incubated for 30 min at 42 C in a hybridization chamber. Fluorescence-labeled probes were dried and resuspended in 20 μl of hybridization solution, which contained 50% formamide, 6x SSPE, 0.5% SDS, 5x Denhardt's solution, blocking solution [2 µg of poly(dA), 4 µg of yeast tRNA, 10 µg of salmon sperm DNA)], 14 µl of master mix solution (70% formamide, 3x Denhardt's solution, 0.7% SDS), and 6 µl of 20x SSPE. The resuspended probes were heated to 100°C for 2 min, vortexed, and collected by a brief spin in a microcentrifuge. The probes were applied to the arrayed surface, covered with a coverslip, and placed in a hybridization chamber overnight at 42°C.
Scan protocol Hybridized slides were washed in each of three solutions (solution 1 is 1x SSC- 0.1% SDS, solution 2 is 2x SSC- 0.1% SDS, and solution 3 is 2x SSC) for 10 min, spun dry, scanned in both Cy3 and Cy5 channels with an Affymetrix 418 Scanner at a 10-µm resolution and a 70% photomultiplier tube value, and exported as 16-bit TIFF images for analysis.
Description NA
Data processing Integrated pixel intensity values for each spot were calculated using TIGR Spotfinder software (TIGR, Rockville, MD; http://www.tigr.org/software) and saved in tab-delimited format for analysis in Mathematica 5.1 (Wolfram Research Inc., Champaign, IL; http://www.wolfram.com). All hybridization data among two sets of dye-swap experiments (representing a total of 4 datasets) were processed according to the following pipeline: (Step 1) Each hybridization dataset was individually processed using a locally weighted linear regression (Lowess) algorithm (smoothing factor = 0.15) to remove systemic dye bias present in each group of spots within a single metarow, metacolumn block of the microarray chip. (Step 2) Following the intra-slide normalization routine described above, all lowess-normalized datasets were loaded simultaneously to rescale each spot through the use of a Z-transformation. Specifically, the global log2 (cy3/cy5) mean and standard deviations were calculated across all four datasets simultaneously and then used to rescale each spot in all four normalized datasets using Equation 1: the rescaled log2 (cy3/cy5) value of an individual spot = [the spot’s own log2 (cy3/cy5) value – global mean of all log2 (cy3/cy5) values] / by the global standard deviation of all log2 (cy3/cy5) values.
 
Submission date Apr 19, 2007
Last update date Apr 19, 2007
Contact name Donald Lee Nuss
E-mail(s) nuss@umbi.umd.edu
Phone 240-314-6218
Fax 240-314-6255
Organization name University of Maryland Biotechnology Institute
Department Center for Biosystems Research
Lab Nuss Lab
Street address 9600 Gudelsky Drive
City Rockville
State/province MD
ZIP/Postal code 20850
Country USA
 
Platform ID GPL4413
Series (2)
GSE7551 Reovirus MyRV2-CpC18 responsive genes in Cryphonectria parasitica
GSE7555 Reoviruses MyRV1-Cp9B21 and MyRV2-CpC18 responsive genes in Cryphonectria parasitica

Data table header descriptions
ID_REF
VALUE Log2 value
C3 Cys3 signal
C5 Cys5 signal
C3 background Cys3 background signal
C5 background Cys5 background signal

Data table
ID_REF VALUE C3 C5 C3 background C5 background
AEST-01-A-02 0.095920422 219355 86574 25920 80208
AEST-01-A-03 -0.57516543 1913218 1459161 47488 112000
AEST-01-A-04 -0.110573847 298246 137555 30275 94458
AEST-01-A-05 -0.746646677 1205074 1036445 41208 111588
AEST-01-A-06 -1.159224016 628900 718039 32436 96036
AEST-01-A-07 0.035200528 1369867 722047 43262 132016
AEST-01-A-08 -0.722693005 815146 836539 37332 93879
AEST-01-A-09 -0.464789173 1689239 1240663 44726 145520
AEST-01-A-10 0.46088062 223980 68844 24511 66802
AEST-01-A-11 1.038156969 2011439 449773 43888 166400
AEST-01-A-12 null
AEST-01-B-01 0.6031194 1071444 377884 30378 87108
AEST-01-B-02 -0.404493959 1923284 1347871 45888 97034
AEST-01-B-03 0.550589263 675965 192769 39096 96768
AEST-01-B-04 0.683073211 353459 124142 36481 81557
AEST-01-B-05 2.000419765 1438221 184424 34980 83528
AEST-01-B-06 0.341711729 865843 380642 46728 110684
AEST-01-B-07 -0.893796056 48141 75147 19872 27738
AEST-01-B-08 -0.437932807 916090 686080 44954 112385
AEST-01-B-09 0.554412878 320225 84041 38247 85690

Total number of rows: 3809

Table truncated, full table size 183 Kbytes.




Supplementary data files not provided

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