|
| Status |
Public on Jul 29, 2014 |
| Title |
Glycolaldehdye-nontreated strain replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Yeast BY4743
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: Haploid
growth condition: anaerobic growth for 4h at 30 ℃
strain: BY4743
treatment: none
|
| Treatment protocol |
Yeast cells were treated with 10 mM of glycolaldehdye for 4 h in static condition
|
| Growth protocol |
For RNA extraction, yeast cells were grown overnight in 2% glucose cntaining synthetic minimal medium with or wothout 10 mM of glycolaldehdye. The, OD600= 0.1 cells were added into the 10 mL of medium and grown at 30℃ for 4 h in static condition, and cells were extracted for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by the hot phenol method (3). After the extraction, RNA was further purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). The purified RNA was stored at -80 °C. For the quality control, RNA purity and integrity were evaluated by OD 260/280 ratio, and analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). RNA extraction and its purification experiments were performed 3 times independently from 3 respective, independent starter cultures
|
| Label |
Cy3-dCTP
|
| Label protocol |
RNA labeling and hybridization were performed by using the Agilent one-color microarray-based gene expression analysis protocol (Agilent Technology, V 6.5, 2010). Briefly, 200 ng of total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The labeled cRNAs were purified by RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 (NanoDrop, Wilmington, USA).
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| |
|
| Hybridization protocol |
Six hundreds nano gram of each labeled cRNA was fragmented by adding 5 μL 10 x blocking agent and 1 μL of 25 x fragmentation buffer, and then heated at 60°C for 30 min. Finally 25 μL 2 x GE hybridization buffer was added to dilute the labeled cRNA. 50 μL of hybridization solution was dispensed into the gasket slide and assembled to the Yeast (V1) Gene Expression Microarray, 4×44K (Agilent®)
|
| Scan protocol |
The slides were incubated for 17 h at 65 °C in an Agilent hybridization oven and washed at room temperature. The hybridized array was immediately scanned with an Agilent Microarray Scanner (Agilent Technologies, Inc.)
|
| Description |
gene expression after 4 h in 2% glucose containing SC medium in the presence of 10 mM glycolaldehdye
|
| Data processing |
Raw data were extracted using the software provided by Agilent Feature Extraction Software (v11.0.1.1). The raw data for same gene was then summarized automatically in Agilent feature extraction protocol to generate raw data text file, providing expression data for each gene probed on the array. Array probes that have Flag A in samples were filtered out. Selected gProcessedSignal value was transformed by logarithm and normalized by quantile method. Statistical significance of the expression data was determined using fold change and independent T-test in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm. For DEG sets, Hierarchical cluster analysis was performed using complete linkage and euclidean distance as a measure of similarity. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using DAVID (http://david.abcc.ncifcrf.gov/home.jsp). All data analysis and visualization of differentially expressed genes was conducted using R 3.0.2 (www.r-project.org)
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| |
|
| Submission date |
Jul 28, 2014 |
| Last update date |
Jul 29, 2014 |
| Contact name |
Lahiru Niroshan Jayakody |
| E-mail(s) |
11975001@edu.cc.saga-u.ac.jp
|
| Phone |
080-3991-8313
|
| Organization name |
Saga University
|
| Department |
Bioscience and Biotechnology
|
| Lab |
Kitagaki laboratory
|
| Street address |
Honjo-machi Honjo
|
| City |
Saga |
| State/province |
Kyushu |
| ZIP/Postal code |
840-8502 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9294 |
| Series (1) |
| GSE59812 |
Gene expression profile of glycolaldehdye-treated yeast cells vs glycolaldehdye-nontreated cells |
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