|
| Status |
Public on Feb 27, 2024 |
| Title |
SWAP_1511_1515_S288vsTAT12 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TAT12
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: TAT12
phase of growth: exponential
|
| Treatment protocol |
All the strains populations were sampled in the middle of exponential phase of growth
|
| Growth protocol |
All the strains were growth in flasks containing defined media with 2% glucose, pH 5.0. Flasks were incubated at 30°C and 250 RPMs of agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted with the YeaStar RNA kit (Zymo Research, Irvine). The RNA was sent to the DNA Microarrays Unit of the Institute of Cellular Physiology, in the National Autonomous University of Mexico for cDNA synthesis, cDNA staining, and hybridization in DNA microarrays.
|
| Label |
Alexa555, Alexa647
|
| Label protocol |
Yeast 50-mer oligo library from MWGBiotech Oligo Sets (http:/www.mwgbiotech.com) were resuspended to 40μM in Micro Spotting solution (Telechem International Inc.). SuperAmine coated slides 25x75 mm (TeleChem International INC) were printed in duplicate, and fixed at 80°C or 4 hours. For pre-hybridization the slides were re-hydrated with water vapor at 60°C, and fixed with two cycles of UV light (1200J). After boiling for two minutes at 92°C, slides were washed with 95% ethanol for one minute and prehybridzed in 5X SSC, 0.1% SDS and 1% BSA for one hour at 42°C .The slides were washed and dried for further hybridization.
|
| |
|
| Channel 2 |
| Source name |
S288c
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: S288c
phase of growth: exponential
|
| Treatment protocol |
All the strains populations were sampled in the middle of exponential phase of growth
|
| Growth protocol |
All the strains were growth in flasks containing defined media with 2% glucose, pH 5.0. Flasks were incubated at 30°C and 250 RPMs of agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted with the YeaStar RNA kit (Zymo Research, Irvine). The RNA was sent to the DNA Microarrays Unit of the Institute of Cellular Physiology, in the National Autonomous University of Mexico for cDNA synthesis, cDNA staining, and hybridization in DNA microarrays.
|
| Label |
Alexa647, Alexa555
|
| Label protocol |
Yeast 50-mer oligo library from MWGBiotech Oligo Sets (http:/www.mwgbiotech.com) were resuspended to 40μM in Micro Spotting solution (Telechem International Inc.). SuperAmine coated slides 25x75 mm (TeleChem International INC) were printed in duplicate, and fixed at 80°C or 4 hours. For pre-hybridization the slides were re-hydrated with water vapor at 60°C, and fixed with two cycles of UV light (1200J). After boiling for two minutes at 92°C, slides were washed with 95% ethanol for one minute and prehybridzed in 5X SSC, 0.1% SDS and 1% BSA for one hour at 42°C .The slides were washed and dried for further hybridization.
|
| |
|
| |
| Hybridization protocol |
10 µg of total RNA were used for cDNA synthesis incorporating dUTP-Alexa555 or dUTP-Alexa647 employing the Firs-Strand cDNA labeling kit (Invitrogen). Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Alexa555 and 650 nm for Alexa647. Equal quantities of labeled cDNA were hybridized using hybridization solution UniHyb (TeleChem International INC). The arrays were incubated for 14 h at 42°C, and then washed tree times with 1X SCC, 0.05 % SDS at room temperature.
|
| Scan protocol |
Acquisition and quantification of array images was performed in GenePix 4100A with its accompanying software GenePix from Molecular Devices. All images were captured using 10µm resolution. For each spot the Alexa555 and Alexa647 density mean value and background mean value were calculated with software ArrayPro Analyzer from Media Cibernetics.
|
| Description |
Gene expression of TAT12 strain compared with S288C under optimal growth conditions
YOMWG_15_11.txt: S288C (Alexa555), TAT12 (Alexa647)
YOMWG_15_15.txt: TAT12 (Alexa555), S288C (Alexa647)
|
| Data processing |
Microarray data analysis was performed with free software genArise, developed in the Computing Unit of Cellular Physiology Institute of UNAM ( http://www.ifc.unam.mx/genarise/ ). GenArise carry out a number of transformations: background correction, lowess normalization, intensity filter, replicates analysis and selecting differentially expressed genes. The goal of genArise is to identify which of the genes show good evidence of being differentially expressed. The software identifies differential expressed genes by calculating an intensity-dependent z-score. Using a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point, and define a z-score where z measures the number of standard deviations a data point is from the mean. zi = (Ri – mean(R)) / sd(R) Where zi is the z-score for each element, Ri is the log-ratio for each element, and sd(R) is the standard deviation of the log-ratio. With this criterion, the elements with a z-score > 2 standard deviations would be the significantly differentially expressed genes. In the Matrix array of processed data, each sample column represent two merged arrays- For example, Sample 1 column represent the merged results of array YOMWG_15_09 and dye-swap array YOMWG_15_10. Each sample column represents Z-scores, and the Ri log-ratio represent TAT12/S288 ratios.
|
| |
|
| Submission date |
Mar 01, 2023 |
| Last update date |
Feb 27, 2024 |
| Contact name |
Luis Caspeta |
| E-mail(s) |
luis.caspeta@ibt.unam.mx
|
| Phone |
7773821619
|
| Organization name |
Instituto de Biotecnología - Universidad Nacional Autónoma de México
|
| Department |
Ingeniería Celular y Biocatálisis
|
| Street address |
Av Universidad 2001
|
| City |
Cuernavaca |
| State/province |
Morelos |
| ZIP/Postal code |
62210 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL33201 |
| Series (1) |
| GSE226362 |
GE changes TTY23, AT22 & TAT12 vs S288C |
|
