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Sample GSM147123 Query DataSets for GSM147123
Status Public on Nov 30, 2006
Title cpst12 deletion mutant (sample B, dye-swap)
Sample type RNA
 
Channel 1
Source name cpst12 deletion mutant
Organism Cryphonectria parasitica
Characteristics cpst12 deletion mutant strain cpst-E1 grown on PDA with cellophane overlay (sample B).
Biomaterial provider Nuss lab
Treatment protocol NA
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 EP155 strain
Organism Cryphonectria parasitica
Characteristics Strain EP155 grown on PDA with cellophane overlay (sample B).
Biomaterial provider Nuss lab
Treatment protocol NA
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 Nov 27, 2006
Last update date Nov 29, 2006
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 (1)
GSE6371 Transcriptional changes caused by cpst12 gene disruption in Cryphonectria parasitica

Data table header descriptions
ID_REF
VALUE Normalized log ratio (refer to Data processing above)
Cy3 cy3 signal
Cy5 cy5 signal
cy3 background cy3 background signal
cy5 background cy5 background signal

Data table
ID_REF VALUE Cy3 Cy5 cy3 background cy5 background
AEST-01-A-02 -0.094007561 408292 101576 21760 17544
AEST-01-A-03 -1.443141394 889065 612773 27918 25344
AEST-01-A-04 1.477178056 654433 84042 16302 19722
AEST-01-A-05 -0.570941318 955895 440804 28000 26800
AEST-01-A-06 -1.789065111 572010 389776 27054 20898
AEST-01-A-07 -1.70459336 755056 526997 26325 23985
AEST-01-A-08 -2.700906434 611211 658309 24420 18796
AEST-01-A-09 -2.631207463 844030 1005378 30600 22400
AEST-01-A-10 0.907905011 615175 102736 23712 24492
AEST-01-A-11 -0.668487185 1417005 673643 28968 27051
AEST-01-A-12 -2.199489804 32341 59581 12709 10224
AEST-01-B-01 -1.702946829 687766 426148 23040 21600
AEST-01-B-02 -0.606072887 2033252 940398 39550 45652
AEST-01-B-03 0.415415599 889615 243794 27738 24522
AEST-01-B-04 0.099151087 644119 170558 33858 26244
AEST-01-B-05 0.855669869 787432 148357 25956 28016
AEST-01-B-06 -1.966239802 1079758 949443 34768 30740
AEST-01-B-07 null
AEST-01-B-08 -1.844978574 832680 718682 32648 27984
AEST-01-B-09 -0.442793278 669138 211783 24108 25872

Total number of rows: 3808

Table truncated, full table size 180 Kbytes.




Supplementary data files not provided

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