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Sample GSM325528 Query DataSets for GSM325528
Status Public on May 21, 2009
Title Yeast_replicate #1_MGD353vsYJR6
Sample type RNA
 
Channel 1
Source name MGD353
Organism Saccharomyces cerevisiae
Characteristics The sample of channel 1 was extracted from total-RNA of Saccharomyces cerevisiae cells. The strain used for this study is MGD353 (wild type striain). This strain was provided by Dr. Brian Rymond's laboratory from the University of Kentucky. Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base).
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from 4 X 10^7 cells derived from five biological replicate cultures of the MGD353 strain using the RNeasy Mini Kit for isolation of total RNA (Qiagen, Hilden, Germany) following manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following manufacturer’s instructions.
Label Cy3
Label protocol Fluorescent cRNA synthesis was produced following the low input fluorescent linear amplification kit from Agilent technologies. As a summary, 1.0 microgram of total was mixed with 2.4 microliters of T7 promoter primer and nuclease free water. The reaction mix was incubated at the 65°C for 10 minutes and then placed the reactions on ice for 5 minutes. 8.5 microliters of cDNA master mix was added to each reaction and incubated at 40°C for two hours. Then the reaction was incubated at 65°C for 15 minutes, to inactivate the reverse transcriptase reaction. Cyanine 3-CTP (10mM) was added to the reaction. 57.6 microliters of transcription master mix was added to the reaction and then incubated at 40°C, to produce the cRNA. The cRNA produced was purified RNeasy mini spin column from Qiagen.

 
Channel 2
Source name YFR23
Organism Saccharomyces cerevisiae
Characteristics YFR23 is chs2::KANMX4, obtained by homologous recombination from the parental haploid wild type (MGD353-46D) using a PCR based method. Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from 4 X 10^7 cells derived from five biological replicate cultures of the YFR23 strain using the RNeasy Mini Kit for isolation of total RNA (Qiagen, Hilden, Germany) following manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following manufacturer’s instructions.
Label Cy5
Label protocol Fluorescent cRNA synthesis was produced following the low input fluorescent linear amplification kit from Agilent technologies. As a summary, 1.0 microgram of total was mixed with 2.4 microliters of T7 promoter primer and nuclease free water. The reaction mix was incubated at the 65°C for 10 minutes and then placed the reactions on ice for 5 minutes. 8.5 microliters of cDNA master mix was added to each reaction and incubated at 40°C for two hours. Then the reaction was incubated at 65°C for 15 minutes, to inactivate the reverse transcriptase reaction. Cyanine 5-CTP (10mM) was added to the reaction. 57.6 microliters of transcription master mix was added to the reaction and then incubated at 40°C, to produce the cRNA. The cRNA produced was purified RNeasy mini spin column from Qiagen.

 
 
Hybridization protocol To each fluorescent cRNA sample, 4 microliters of 25X fragmentation buffer was added, then incubated at 60°C for 30 minutes. After the incubation, 100 microliters of 2X hybridization buffer were added to the sample. Samples were placed into the array slides. The array slides were placed in the hybridization chambers and incubated at 60°C for 17 hours. Array slides were washed in Wash solution 1(6X SSPE, 0.005%N-Lauroylsarcosine), Wash solution 2 (0.06X SSPE, 0.005%N-Lauroylsarcosine) and finally acetonitrile as stabilization and drying solution.
Scan protocol Arrays were scanned in versarray scanner (from Biorad) with a detector sensitiity of 800 and laser power of 85% with a resolution of 5 micrometers. The images were saved and pre-processed with the Imagene 3.0 software (Biodiscovery) to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 text files (microarray raw data).
Description The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change (VALUE) after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.01 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma.
Data processing The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change (VALUE) after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.01 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma.
 
Submission date Sep 30, 2008
Last update date May 21, 2009
Contact name José R Rodríguez-Medina
E-mail(s) jorrodriguez@rcm.upr.edu
Organization name University of Puerto Rico Medical Sciences Campus
Department Biochemistry
Lab A-633
Street address PO BOX 365067
City San Juan
State/province PR
ZIP/Postal code 00936-5067
Country USA
 
Platform ID GPL2883
Series (1)
GSE12994 Signature gene expression profiles for cytokinesis mutants in the budding yeast Saccharomyces cerevisiae

Data table header descriptions
ID_REF
VALUE log ratio for normalized signal intensities for chs2 versus wild type (log fold change)

Data table
ID_REF VALUE
1 -2.107375446
2 -1.619835408
3 0.048152381
4 -0.610696353
5 0.457230275
6 -0.039519051
7 -0.029639342
8 0.231942545
9 0.023979224
10 0.517309813
11 -0.075687757
12 -0.500102913
13 0.929312984
14 -0.563565528
15 -0.775972685
16 -0.675833934
17 -0.962099675
18 0.207963367
19 -0.272744111
20 -0.212443204

Total number of rows: 10807

Table truncated, full table size 183 Kbytes.




Supplementary file Size Download File type/resource
GSM325528_A2012_MGDcy3t_1.txt.gz 1.4 Mb (ftp)(http) TXT
GSM325528_A2012_YFR23cy5t_2.txt.gz 1.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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