|
| Status |
Public on Mar 01, 2017 |
| Title |
strain ITD00185, time point 48h, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
strain ITD00185, time point 48h, replicate 2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: ITD00185
time point: 48 h
|
| Growth protocol |
Fermentations in synthetic agave must at 28 ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction method was based on consecutive treatments with phenol-tris, phenol-chloroform (5:1) and chloroform-isoamyl alcohol (24:1) and a final precipitation with ethanol and sodium acetate (Garcia-Martinez et al., 2004).
|
| Label |
Cy5
|
| Label protocol |
2-4 ug of total RNA was linearly amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies™, Ca, USA). 2-3 ug of amplified cRNA was used as template for cDNA synthesis. cDNA was marked indirectly with “SuperScript™ Indirect cDNA Labeling System” (Invitrogen™, SanDiego, CA).
The fluorophores used were Cy3 and Cy5 mono-reactiveDye (Amersham GE Healthcare™, Amersham UK) and dye incorporation was monitored by a Nanodrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
sample mix
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: mixed
time point: 48 h
|
| Growth protocol |
Fermentations in synthetic agave must at 28 ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction method was based on consecutive treatments with phenol-tris, phenol-chloroform (5:1) and chloroform-isoamyl alcohol (24:1) and a final precipitation with ethanol and sodium acetate (Garcia-Martinez et al., 2004).
|
| Label |
Cy3
|
| Label protocol |
2-4 ug of total RNA was linearly amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies™, Ca, USA). 2-3 ug of amplified cRNA was used as template for cDNA synthesis. cDNA was marked indirectly with “SuperScript™ Indirect cDNA Labeling System” (Invitrogen™, SanDiego, CA).
The fluorophores used were Cy3 and Cy5 mono-reactiveDye (Amersham GE Healthcare™, Amersham UK) and dye incorporation was monitored by a Nanodrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
A mixture of 200 to 300 pmol of the two samples labelled was concentrated in a Concentrator Plus (Eppendorf™, Hamburg, Germany). Competitive hybridization was performed in hybridization chambers AHC (ArrayIt Corporation, CA, USA) at 42°C overnight. (Prehybridization solution contained 3X SSC, 0.1% SDS and 0.1 mg/ml BSA; hybridization solution
contained 5X SSC, 0.1% SDS and 0.1 mg/ml of salmon DNA. Microarrays were washed manually with different solutions containing different SSC 20X and SDS 10% concentrations (Sol.1: 2X SSC-0.1% SDS; Sol.2: 0.1X SSC-0.1% SDS; Sol.3: 0.1 SSC; Sol4: 0.01X SSC).
|
| Scan protocol |
Signal intensities of Cy3 and Cy5 were acquired with an Axon GenePix 4100A scanner (Molecular devices, CA, USA) using GenePix Pro v.6.1 software, at a resolution of 10 μm.
|
| Description |
Biological replicate two of three
|
| Data processing |
Raw data with a global background subtraction were generated from GenePix pro 6.0. Analyses were done using Acuity 4.0 software (Molecular Devices, CA, USA).The individual data sets were normalized to a log2 ratio value of 1. After lowess normalization, data were filtered to remove spots flagged as not found.
Only spots with at least two replicates were considered. Finally, replicates were combined and their medians were calculated.
Genes with a two-fold log2 ratio values were considered to be significantly expressed.
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| |
|
| Submission date |
Feb 28, 2017 |
| Last update date |
Mar 01, 2017 |
| Contact name |
Roberto Pérez |
| E-mail(s) |
rober@iata.csic.es
|
| Phone |
0034963900022
|
| Organization name |
Centro Superior de Investigaciones Científicas
|
| Street address |
Av Agustín Escardino 7
|
| City |
Paterna |
| State/province |
Valencia |
| ZIP/Postal code |
46980 |
| Country |
Spain |
| |
|
| Platform ID |
GPL23130 |
| Series (1) |
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