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Sample GSM182832 Query DataSets for GSM182832
Status Public on Apr 20, 2007
Title Reovirus MyRV1-Cp9B21 infection (sample A, dye-swab)
Sample type RNA
 
Channel 1
Source name Wild type strains EP155
Organism Cryphonectria parasitica
Characteristics Strain EP155 grown on PDA with cellophane overlay (sample A).
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 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 infected with reovirus MyRV1-Cp9B21
Organism Cryphonectria parasitica
Characteristics Reovirus MyRV1-Cp9B21 infected strain EP155 grown on PDA with cellophane overlay(sample A).
Biomaterial provider Hillman lab
Treatment protocol Reovirus MyRV1-Cp9B21 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 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 18, 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)
GSE7546 Reovirus MyRV1-Cp9B21 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
VALUE Normalized log ratio (INV-VALUE, refer to Data processing above)

Data table
ID_REF Value C3 C5 C3 background C5 background VALUE
AEST-01-A-02 null
AEST-01-A-03 -0.248716559 1157071 198298 29190 19530 0.248716559
AEST-01-A-04 0.215376298 150719 16555 19320 13020 -0.215376298
AEST-01-A-05 0.856271126 445409 32132 24613 12388 -0.856271126
AEST-01-A-06 0.931849032 150364 4465 18769 12330 -0.931849032
AEST-01-A-07 -2.999149477 72907 267040 16761 9879 2.999149477
AEST-01-A-08 null
AEST-01-A-09 -0.258946664 1249383 235379 24875 16517 0.258946664
AEST-01-A-10 -0.778075371 1067739 292210 26058 17574 0.778075371
AEST-01-A-11 null
AEST-01-A-12 null
AEST-01-B-01 0.835420497 309476 21401 17420 14820 -0.835420497
AEST-01-B-02 -0.427584643 183416 49050 29484 18333 0.427584643
AEST-01-B-03 null
AEST-01-B-04 1.348420379 25032 2307 10008 6624 -1.348420379
AEST-01-B-05 -0.809479222 138493 61491 33674 17628 0.809479222
AEST-01-B-06 null
AEST-01-B-07 0.356734914 391098 41271 32665 19270 -0.356734914
AEST-01-B-08 0.263357301 471688 61513 36456 18848 -0.263357301
AEST-01-B-09 0.523470539 172311 12502 22044 16366 -0.523470539

Total number of rows: 3565

Table truncated, full table size 194 Kbytes.




Supplementary data files not provided

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