|
| Status |
Public on Oct 29, 2015 |
| Title |
pH 6.1 vs pH7_summarization of 3 technical replicates |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chemostat cultures cells
|
| Organism |
Sinorhizobium meliloti |
| Characteristics |
strain: 1021
|
| Treatment protocol |
pH 7.0
|
| Growth protocol |
Cells growth at constant Dilution rate and controlled pH 7.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the RNeasy mini kit (QIAGEN, Hildesheim, Germany). By ribolysation (30 s; speed, 6.5; Hybaid, Heidelberg, Germany) cells were disrupted in the RLT buffer provided with the kit in Fast Protein Tubes (Qbiogene, Carlsbad, CA).
|
| Label |
Cy3
|
| Label protocol |
Fluorescent-labeled cDNA was prepared according to an amino-allyl dye coupling protocol. Starting from 10 to 20 μg total RNA amino-allyl modified first strand cDNA was synthesized by reverse transcription using random hexamer primers, SuperScriptII reverse transcriptase and a dNTP + aa-dUTP mixture. After hydrolysis and cleanup using Microcon-30 filters the N-hydroxysuccinimidyl ester dyes were coupled to the amino-allyl-labeled first-strand cDNA. Uncoupled dye was removed using QiaQuick PCR Purification columns.
|
| |
|
| Channel 2 |
| Source name |
Chemostat cultures cells
|
| Organism |
Sinorhizobium meliloti |
| Characteristics |
strain: 1021
|
| Treatment protocol |
pH 6.1
|
| Growth protocol |
Cells growth at constant Dilution rate and controlled pH 6.1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the RNeasy mini kit (QIAGEN, Hildesheim, Germany). By ribolysation (30 s; speed, 6.5; Hybaid, Heidelberg, Germany) cells were disrupted in the RLT buffer provided with the kit in Fast Protein Tubes (Qbiogene, Carlsbad, CA).
|
| Label |
Cy5
|
| Label protocol |
Fluorescent-labeled cDNA was prepared according to an amino-allyl dye coupling protocol. Starting from 10 to 20 μg total RNA amino-allyl modified first strand cDNA was synthesized by reverse transcription using random hexamer primers, SuperScriptII reverse transcriptase and a dNTP + aa-dUTP mixture. After hydrolysis and cleanup using Microcon-30 filters the N-hydroxysuccinimidyl ester dyes were coupled to the amino-allyl-labeled first-strand cDNA. Uncoupled dye was removed using QiaQuick PCR Purification columns.
|
| |
|
| |
| Hybridization protocol |
Microarrays were prehybridized for 45 min at 42 °C in Easyhyb hybridization solution supplemented with 5 μg/ml sonicated salmon sperm DNA. Following prehybridization microarrays were washed in Milli-Q water (21 °C, 1 min), dunked in ethanol (21 °C, 10 s) and centrifuged (185×g, 3 min, 20 °C). Hybridization was performed at 42 °C for 16 h in Easyhyb hybridization solution supplemented with 50 μg/ml sonicated salmon sperm DNA in a final volume of 65 μl under a cover slip. Before applying the hybridization sample to the microarray, it was denatured for 5 min at 65 °C. Microarrays were washed once in 2× SSC, 0.2% SDS (5 min, 42 °C), twice in 0.2× SSC, 0.1% SDS (2 min, 21 °C) and twice in 0.2× SSC (2 min, 21 °C)
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
Images were quantified using Agilent Feature Extraction Software (version A.7.5).
|
| Description |
Summarization of 3 Technical Replicates with 3 internal replicates each
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used. Data have been summarized across 3 technical replicates and 3 internal replicates on each array. Processed data file includes M-value (log2 fold-change Cy5/Cy3 representing pH6.1/pH7), P-value and A-value.
Data analysis was carried out according to Ruberg et al, 2003 (PMID 14651866). Briefly,genes were regarded as differentially expressed if P ≤ 0.05, M-values ≥ 1 or ≤ −1 and A-values ≥ 9.0. Normalization and t-statistics were carried out using the EMMA 1.0 microarray data analysis software developed at the Center for Genome Research at Bielefeld University (http://emma.cebitec.uni-bielefeld.de).
|
| |
|
| Submission date |
Oct 28, 2015 |
| Last update date |
Oct 30, 2015 |
| Contact name |
Antonio Lagares |
| Organization name |
IBBM
|
| Department |
Ciencias Biológicas
|
| Street address |
49 y 115
|
| City |
La Plata |
| State/province |
Bs As |
| ZIP/Postal code |
1900 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL8499 |
| Series (1) |
| GSE74449 |
A consolidated analysis of the physiological and molecular responses induced under acid stress in the legume symbiont model soil bacterium Sinorhizobium meliloti |
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