|
| Status |
Public on Nov 27, 2012 |
| Title |
JP1 strain T6h [T6h- 1] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
JP1 strain T6h
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: diploid homothallic yeast isolated from Japungu Agroindustrial (Santa Rita, PB)
developmental stage: T6h fermetaion stage
|
| Treatment protocol |
Yeast cells were cultivated in YPD medium (20 g/l glucose, 20 g/l yeast extract and 10 g/l peptone) at 33ºC and 130 rpm in a rotatory shaker in batches of 24 h. After this period, yeast cells were recovered and suspended in fresh medium for a new batch. This procedure was repeated until reaching enough biomass for the fermentations assays.
|
| 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. For comparison among industrial strains, dye-swap procedure was used for labelling the same sample with Cy3 or Cy5 CTP.
|
| |
|
| Channel 2 |
| Source name |
Lab strain BY4741
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Lab strain BY4741 (EUROSCARF collection)
developmental stage: T0h fermetaion stage
|
| Treatment protocol |
Yeast cells were cultivated in YPD medium (20 g/l glucose, 20 g/l yeast extract and 10 g/l peptone) at 33ºC and 130 rpm in a rotatory shaker in batches of 24 h. After this period, yeast cells were recovered and suspended in fresh medium for a new batch. This procedure was repeated until reaching enough biomass for the fermentations assays.
|
| 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. For comparison among industrial strains, dye-swap procedure was used for labelling the same sample with Cy3 or Cy5 CTP.
|
| |
|
| |
| Hybridization protocol |
RNA from lab strain BY4741 was labelled with Cy3 and mixed with Cy5-labelled RNA from industrial strains. 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 [20] 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 [20]. Groups of genes with statistical significance (Adj.p<0.05) were utilized.
|
| |
|
| Submission date |
Nov 21, 2012 |
| Last update date |
Nov 27, 2012 |
| Contact name |
Marcos Antonio Morais |
| E-mail(s) |
marcos.morais@pesquisador.cnpq.br
|
| Organization name |
Federal University of Pernambuco
|
| Department |
Department of Genetics
|
| Lab |
Interdepartmental Research Group in Metabolic Engineering
|
| Street address |
Av. Moraes Rego, 1235
|
| City |
Recife |
| State/province |
Pernambuco |
| ZIP/Postal code |
50670-901 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE42433 |
Yeast transcriptomics in sugarcane-based ethanol fermentation |
|
