|
| Status |
Public on Jan 26, 2007 |
| Title |
S._cerevisiae_RM11-1a/BY4741_OD600=1_8-replicates-dye-swapping |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RM11-1a
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
S. cerevisiae RM11-1a strain
|
| Treatment protocol |
S. cerevisiae RM11-1a strain was grown to OD600=1 with YPAD media.
|
| Growth protocol |
S. cerevisiae RM11-1a strain was grown to OD600=1 with YPAD media.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The yeast cells were harvested at the OD600=1.0 and the total RNAs were extracted by the hot acid phenol-chloroform method.
The mRNA was isolated by using the QIAGEN Oligotex mRNA purification kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
cy5/cy3
|
| Label protocol |
amino-allyl dye coupling procedure
1. 1 μl 0.5 M NaHCO3, pH 9.0 + 9 μl Aminoallyl-cDNA solution (final 50 mM)
2. Transfer 1. to microfuge tube containing dried down dye**
3. Pipet up and down
4. R/T 1 hr in the dark
5. Mix and spin every 15 min
|
| |
|
| Channel 2 |
| Source name |
BY4741
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
S. cerevisiae BY4741 strain
|
| Treatment protocol |
S. cerevisiae BY4741 strain was grown to OD600=1 with YPAD media.
|
| Growth protocol |
S. cerevisiae BY4741 strain was grown to OD600=1 with YPAD media.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The yeast cells were harvested at the OD600=1.0 and the total RNAs were extracted by the hot acid phenol-chloroform method.
The mRNA was isolated by using the QIAGEN Oligotex mRNA purification kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
cy3/cy5
|
| Label protocol |
amino-allyl dye coupling procedure
1. 1 μl 0.5 M NaHCO3, pH 9.0 + 9 μl Aminoallyl-cDNA solution (final 50 mM)
2. Transfer 1. to microfuge tube containing dried down dye**
3. Pipet up and down
4. R/T 1 hr in the dark
5. Mix and spin every 15 min
|
| |
|
| |
| Hybridization protocol |
The hybridization of cDNA samples to the probes were done with the MAUI hybridization System (Biomicro) at 42oC for 16 hours.
Pre-hybridization
1. Incubate arrays in pre-hybridization buffer, 42℃, 30-60 min
Final conc. Volume
5X SSC 12.5 ml
0.1% SDS 0.5 ml
0.1 mg/ml BSA 0.5 ml
ddH2O 36.5 ml
Total 50 ml
2. Wash array by immersing in water
3. Dry by centrifugation ~ 5 min
Hybridization
1. Prepare probe in fresh hybridization solution in 0.5 ml tube
Final conc. Volume
Labeled cDNA 42 μl
5X SSC 15 μl
0.1% SDS 3 μl
Total 60 μl
2. denature the probe at 98℃ for 3 min, pop-spin, and cool to R/T
3. Place in hybridization chamber
4. Hybridize in 42℃ hybridizer (MAUI hybridization system) for 16 hr
Post-hybridization (Washing)
(Pre-warm the washing buffers in 30℃)
1. Dissemble the hybridization chamber
2. Immerse in 30℃-prewarmed (2X SSC+0.1% SDS), peel off the coverslip
3. Wash in 30℃-prewarmed (2X SSC+0.1% SDS) in R/T, 5 min
4. Wash in 30℃-prewarmed (0.1X SSC, 0.1% SDS) in R/T, 5 min
5. Wash in 30℃-prewarmed 0.1X SSC, in R/T, 5 min
6. Dry by centrifuge, scan
|
| Scan protocol |
The microarray was scanned with GenePix 4000B microarray scanner (Axon Instruments) with the GenePix 5 software package.
|
| Description |
We could like to know the expression divergency of S. cerevisiae strains RM11-1a and BY4741.
Yeast strains were grown in YPAD and harvested at the mid-log phrase. Overnight yeast cultures were used to prepare the starting cultures with OD600=0.1 and were grown in YPAD media at 30oC with 250 rpm shaking. The yeast cells were harvested at the OD600=1.0 and the total RNAs were extracted by the hot acid phenol-chloroform method.
|
| Data processing |
See linked supplementary file: GSM157844_Data_processing.pdf
|
| |
|
| Submission date |
Jan 23, 2007 |
| Last update date |
Jan 25, 2007 |
| Contact name |
Huang-Mo Sung |
| E-mail(s) |
hmsung@gmail.com
|
| Phone |
886-2-27898756
|
| Organization name |
Academia Sinica, Taiwan
|
| Department |
Genomics Research Center
|
| Lab |
Dr. Wen-Hsiung Li
|
| Street address |
128 Sec.2 Academia Rd.
|
| City |
Taipei |
| ZIP/Postal code |
115 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL4773 |
| Series (1) |
| GSE6848 |
Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors |
|
