|
| Status |
Public on Mar 12, 2014 |
| Title |
slt2 2.5/7 A and B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
slt2 2.5 A and B
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Lab strain BY4741 (EUROSCARF collection)
developmental stage: stress YPD pH 2.5 - sulfuric acid
|
| Treatment protocol |
The cultures were centrifuged and each one was re-suspended with YPD medium pH 2.5 adjusted with sulfuric acid. After incubation for 1 h at 30°C with orbital agitation (150 rpm), the cells were recovered by centrifugation and immediately used to RNA extraction
|
| Growth protocol |
Cell from BY4741 strain (EUROSCARF collection) and their derivate mutants hog1∆ and slt2∆ were growth overnight and were transferred to fresh neutral YPD medium (20 g/l glucose, 20 g/l yeast extract and 10 g/l peptone) at 30ºC and 150 rpm in a rotatory shaker at an initial cell density of OD600 0.1 and cultivated to OD600 0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA in the lysates was purified by using RNAspin Mini RNA Isolation Kit (cat. nr. 25-0500-71, GE HealthCare, USA) following manufacturer´s instructions and suspended in 25 μl nuclease-free water.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of cDNA and labelling was performed by Two-Color Low Input Quick Amp Labeling Kit (cat. nr. 5190-2306, Agilent, USA), with Two-Color RNA spike-in kit for internal control (cat. nr. 5188-5279, Agilent, USA). For yeast samples, 100 ng of purified RNA was used for labelling.
|
| |
|
| Channel 2 |
| Source name |
slt2 7 A and B
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Lab strain BY4741 (EUROSCARF collection)
developmental stage: stress YPD pH 2.5 - sulfuric acid
|
| Treatment protocol |
The cultures were centrifuged and each one was re-suspended with YPD medium pH 2.5 adjusted with sulfuric acid. After incubation for 1 h at 30°C with orbital agitation (150 rpm), the cells were recovered by centrifugation and immediately used to RNA extraction
|
| Growth protocol |
Cell from BY4741 strain (EUROSCARF collection) and their derivate mutants hog1∆ and slt2∆ were growth overnight and were transferred to fresh neutral YPD medium (20 g/l glucose, 20 g/l yeast extract and 10 g/l peptone) at 30ºC and 150 rpm in a rotatory shaker at an initial cell density of OD600 0.1 and cultivated to OD600 0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA in the lysates was purified by using RNAspin Mini RNA Isolation Kit (cat. nr. 25-0500-71, GE HealthCare, USA) following manufacturer´s instructions and suspended in 25 μl nuclease-free water.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of cDNA and labelling was performed by Two-Color Low Input Quick Amp Labeling Kit (cat. nr. 5190-2306, Agilent, USA), with Two-Color RNA spike-in kit for internal control (cat. nr. 5188-5279, Agilent, USA). For yeast samples, 100 ng of purified RNA was used for labelling.
|
| |
|
| |
| Hybridization protocol |
RNA from lab strains BY4741, hog1 and slt2 mutants that had grown in YPD medium pH 7, were labelled with Cy3 and mixed with Cy5-labelled RNA from BY4741, hog1 and slt2 mutants that had grown in YPD medium pH 2.5. Target and reference cRNA samples were mixed and the hybridizations were performed on yeast gene expression 8x15K spots slides (cat. nr. G4813A-016322, Agilent, USA) at 65 ºC for 17 hours at 10 r.pm. in the Microarray Hybridization Oven (cat. nr. G2545A, Agilent, USA).
|
| Scan protocol |
The slides were placed into the High-Resolution Microarray Scanner (cat. nr. G2505-60502, Agilent, USA). The fluorescence data were extracted (.txt files) for each spot by using Scan Control 8.5.1 software (Agilent, USA)
|
| Data processing |
Data were then analyzed using specific packages from the R statistical language with the specific LIMMA and MArray packages. Background correction was done with normexp method. Robust spline and quantile methods were used for normalization within and between arrays, respectively. A list of differentially expressed genes was generated by applying adjusted p-values for multiple tests. Groups of genes with statistical significance (Adj.p<0.05) were utilized.
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| |
|
| Submission date |
Mar 11, 2014 |
| Last update date |
Mar 12, 2014 |
| Contact name |
Rodrigo Mendonça Lucena |
| E-mail(s) |
lucenarm2@gmail.com
|
| Organization name |
Federal University of Pernambuco
|
| Department |
Genetics
|
| Lab |
Interdepartmental Research Group in Metabolic Engineering
|
| Street address |
Av. Jorn. Aníbal Fernandes
|
| City |
Recife |
| State/province |
Pernambuco |
| ZIP/Postal code |
50740-560 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE51353 |
The role of the Hog1 and Slt2 MAP Kinases in the regulation of Saccharomyces cerevisiae gene expression upon stress by sulfuric acid |
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