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Sample GSM249128 Query DataSets for GSM249128
Status Public on Sep 06, 2008
Title msn2/4 mutant 3 hours exposure to zymolyase 2
Sample type RNA
 
Channel 1
Source name total RNA Saccharomyces cereviase msn2/4 mutant, 3 hours exposure to zymolyase
Organism Saccharomyces cerevisiae
Characteristics S. cerevisiae (msn2/4 mutant strain BY4741) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with Zymolyase to a final concentration of 0,8U/ml. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from exponentially growing cells (5 x 108) by the “mechanical disruption protocol” using the RNeasy MIDI kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label Cy5
Label protocol cDNA was synthesized from 25–30 μg of total RNA by reverse transcription, using the CyScribeTM First-Strand cDNA Labeling Kit, incorporating Cy5- dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. Labeled cDNA was dried in a vacuum trap and used as a hybridization probe after resuspension in 45 μl of hybridization solution (50% Formamide, 6 x SSC, 0.5% SDS, 5 x Denhardt’s, 20 μg of poly(A) (P-9403, Sigma) and salmon sperm (Invitrogen, 100 μg/ml)
 
Channel 2
Source name total RNA Saccharomyces cereviase msn2/4 mutant, without zymolyase
Organism Saccharomyces cerevisiae
Characteristics S. cerevisiae (msn2/4 mutant strain BY4741) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with Zymolyase to a final concentration of 0,8U/ml. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from exponentially growing cells (5 x 108) by the “mechanical disruption protocol” using the RNeasy MIDI kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label Cy3
Label protocol cDNA was synthesized from 25–30 μg of total RNA by reverse transcription, using the CyScribeTM First-Strand cDNA Labeling Kit, incorporating Cy3-dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. Labeled cDNA was dried in a vacuum trap and used as a hybridization probe after resuspension in 45 μl of hybridization solution (50% Formamide, 6 x SSC, 0.5% SDS, 5 x Denhardt’s, 20 μg of poly(A) (P-9403, Sigma) and salmon sperm (Invitrogen, 100 μg/ml)
 
 
Hybridization protocol Slides were prehybridized in prehybridization
solution (6 x SSC, 0.5% SDS, 1% bovine serum albumin (A-7906,
Sigma) for 1 h and were then hybridized overnight in a LucideaTM SlidePro Automated Hybridization Station (Amersham Biosciences). Before scanning, the chips were washed and dried in a same LucideaTM SlidePro Automated Hybridization
Scan protocol Microarrays were scanned with aGenePix 4000B scanner (Axon Instruments, Union City, CA) at a resolution
of 5 μm (PMT values ranging from 550 to 700 and laser
power 100%). GenePix Pro 4.0 analysis software (Axon Instruments, Union City, CA) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to the
manufacturer’s instructions.
Description For each condition tested, comparison of treated and non-treated samples, the total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.
Data processing Flagged spots and spots with an average intensity minus background below the mean of the background (considering this as the median of the local background for each spot) for all the non-flagged spots in any of the channels (Cy3 or Cy5) were not retained for further analysis. Within this group, the spots showing in one channel a value of intensity minus background higher than 5 times the mean of the background for all spots in the microarray for that channel were recovered. The reproducibility of replicates in each microarray (two spots per ORF) was analyzed by creating a normal distribution log2 (RA _ 1/RB), where RA and RB are the ratios of replicated spots after background subtraction and normalization. Replicates that exceeded the average by more than _3 S.D. were discarded. Data analysis was accomplished using the GeneSight 4.0 software package (BioDiscovery Inc, El Segundo, CA). Locally weighed linear regression (lowess) analysis was performed as a normalization method to remove the intensity-dependent deviation in the log2(ratio) values. Significance analysis of the results was conducted using a Student’s t test (GeneSight). Genes with p values of less than 0.05 were considered to be significantly differentially expressed with respect to the treatment condition.
 
Submission date Dec 12, 2007
Last update date Aug 14, 2011
Contact name Javier Arroyo
E-mail(s) [email protected], [email protected]
Phone 0034-913941746
URL http://ucm.es/info/mfar
Organization name Facultad de Farmacia (UCM)
Department Microbiologia II
Lab U4-Javier Arroyo
Street address Plaza Ramon y Cajal S/N
City Madrid
State/province Madrid
ZIP/Postal code 28040
Country Spain
 
Platform ID GPL7054
Series (2)
GSE9934 msn2/4 mutant 3 hour exposure to zymolyase
GSE12684 hog1, slt2, rlm1, and msn2/4 mutants exposed to zymolyase

Data table header descriptions
ID_REF
CH1_SIG-CH1_BKD Normalized Intensity - Background for channel 1
CH2_SIG-CH1_BKD Normalized Intensity - Background for channel 2
VALUE lowess normalized log2 of test/reference
PRE_VALUE Normalized ratio (CH2_SIG-CH2_BKD/CH1_SIG-CH1_BKD)
INV_VALUE log2 of PRE_VALUE

Data table
ID_REF CH1_SIG-CH1_BKD CH2_SIG-CH1_BKD VALUE PRE_VALUE INV_VALUE
YAL001C
YAL002W 1586 1539.5675 0.0429 0.9707 -0.0429
YAL003W 12534.5 20694.3644 -0.7233 1.651 0.7233
YAL004W
YAL005C 52327 39850.1508 0.3931 0.7615 -0.3931
YAL007C 1530 1637.0938 -0.0976 1.07 0.0976
YAL008W 540.5 462.2109 0.226 0.855 -0.2260
YAL009W 142.5 131.0184 0.1641 0.8925 -0.1641
YAL010C
YAL011W 242 235.5881 0.0368 0.9748 -0.0368
YAL012W 5259 7961.407 -0.5976 1.5132 0.5976
YAL013W 299.5 273.135 0.1296 0.9141 -0.1296
YAL014C 640 666.1193 -0.0596 1.0422 0.0596
YAL015C 136.5 233.174 -0.7717 1.7073 0.7717
YAL016W 1369 690.934 0.9842 0.5055 -0.9842
YAL017W 2143 1147.8861 0.9021 0.5351 -0.9021
YAL018C
YAL019W 325.5 182.0578 0.838 0.5594 -0.8380
YAL020C 187.5 331.632 -0.8221 1.768 0.8221
YAL021C 408 199.2257 1.0374 0.4872 -1.0374

Total number of rows: 6220

Table truncated, full table size 169 Kbytes.




Supplementary file Size Download File type/resource
GSM249128.gpr.gz 468.9 Kb (ftp)(http) GPR
Processed data included within Sample table

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