|
| Status |
Public on Dec 31, 2008 |
| Title |
msg5 f.alpha |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
msg5
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
msg5 strain (control condition)
|
| Biomaterial provider |
Marta Flández
|
| Treatment protocol |
growth at conditions 24 °C 200rpm
|
| Growth protocol |
msg5 delta strain was grown on YPD 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 4h. At this point time 0 begins. Cells were collected after 90 minutes of growth, frozen at -80 °C and processed for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Saccharomyces cerevisiae total RNA was extracted by mechanical disruption following the instructions of RNeasy Midi kit manufacturer (QIAGEN). Total concentration of RNA obtained was measured at 260 nm and sample quality was checked using RNA Nano Labchips in a Bioanalyzer 2100B (Agilent Technologies, Palo Alto, CA). cDNA synthesis and incorporation of aminoallyl nucleotides is carried out by retrotranscription from 25-30 microgrames of total RNA using Amersham Bioscienceskit .
|
| Label |
Cy3, Cy5
|
| Label protocol |
Labelling with Cy3 or Cy5 groups of the corresponding cDNA was achieved using CyScribeTM Post-Labelling kit (Amersham Biosciences) incubating the samples in the dark for two hours. Fluorocrome incorporation was measured using espectophotometer.
|
| |
|
| Channel 2 |
| Source name |
msg5 factor alpha 90 min
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
msg5 strain factor alpha 50 mM (experimental condition)
|
| Biomaterial provider |
Marta Flández
|
| Treatment protocol |
growth at conditions 24 °C 200rpm
|
| Growth protocol |
msg5 delta strain was grown on YPD 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 4h. At this point time 0 begins. Cells were collected after 90 minutes of growth under treatment with 50mM of factor alpha , frozen at -80 °C and processed for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Saccharomyces cerevisiae total RNA was extracted by mechanical disruption following the instructions of RNeasy Midi kit manufacturer (QIAGEN). Total concentration of RNA obtained was measured at 260 nm and sample quality was checked using RNA Nano Labchips in a Bioanalyzer 2100B (Agilent Technologies, Palo Alto, CA). cDNA synthesis and incorporation of aminoallyl nucleotides is carried out by retrotranscription from 25-30 microgrames of total RNA using Amersham Bioscienceskit .
|
| Label |
Cy3, Cy5
|
| Label protocol |
Labelling with Cy3 or Cy5 groups of the corresponding cDNA was achieved using CyScribeTM Post-Labelling kit (Amersham Biosciences) incubating the samples in the dark for two hours. Fluorocrome incorporation was measured using espectophotometer.
|
| |
|
| |
| Hybridization protocol |
Treated and control cDNA labeled with different dyes are mixed, dried in speed vac and resuspended in hybridation solution: 50% Formamide, 6x SSC, 0.5% SDS, 5x Denhardt´s, 20 microgrammes PolyA (Sigma) and salmon sperm (Gibco) 100 microgrammes/ml.
Hybridation of slides with labeled cDNA is carried out for 18 hours at 42ºC in a fluidic station LucideaTM SlidePro (Amersham Biosciences) followed by several washes and drying of slides.
|
| Scan protocol |
Microarrays were scanned using GenePix 4000B scanner (Axon Instruments) at 5 micron/píxel, using laser excitation between 550 and 700 PMT and 100% laser power.
|
| Description |
Image analysis and data segmentation were done using GenePix Pro software 5.0 (Axon Instruments) obtaining a txt file to proceed with the data analysis.
|
| Data processing |
Filtering of low neat intensity and inconsistent spot data was followed by Lowess normalization and calculation of ratios. Statistical t-student test was carried out to determine the significance of the induction and repression ratios.
The Data table represents results condensed from four microarrays (coming from two different biological samples labeled using dye swapping strategy).
|
| |
|
| Submission date |
Jul 18, 2008 |
| Last update date |
Nov 27, 2008 |
| Contact name |
Maria Molina |
| Organization name |
Universidad Complutense de Madrid
|
| Department |
Microbiologia II Fcc Farmacia
|
| Lab |
U3
|
| Street address |
Plaza Ramon y Cajal s/n
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28040 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7054 |
| Series (1) |
| GSE12104 |
Transcriptional profiles of msg5 mutants and msg5 mutants in the presence/absence of alpha factor |
|
