|
| Status |
Public on Sep 18, 2008 |
| Title |
Yeast_myo1_replicate#1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cy3_YJR12 (wild type)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
The sample of channel 1 was extracted from total-RNA of Saccharomyces cerevisiae cells. YJR12 (wild type) strain was obtained as haploid segregant from a cross between YJR6(myo1) and BY4741 (wild type, obtained from ATTC). 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 triplicate biological replicate cultures of the YJR12 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 |
Cy5_YJR13(myo1 delta mutant)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
The sample of channel 2 was extracted from total-RNA of Saccharomyces cerevisiae cells. YJR13 (myo1∆) strain was obtained as haploid segregant from a cross between YJR6 (myo1∆) and BY4741 (wild type,obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in histidine dropout media (CSM-HIS-,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 triplicate biological replicate cultures of the YJR13 strain using the RNeasy Mini Kit for isolation of total RNA (Qiagen) 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 28, 2006 |
| Last update date |
Sep 23, 2008 |
| 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 |
GPL884 |
| Series (1) |
| GSE5931 |
Global mRNA expression analysis in myo1 delta strains of the budding yeast Saccharomyces cerevisiae |
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