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Status |
Public on Oct 23, 2014 |
Title |
Control K.vulgare Cell Replicate 3 |
Sample type |
RNA |
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Channel 1 |
Source name |
K.vulgare control cells
|
Organism |
Ketogulonicigenium vulgare |
Characteristics |
strain: WSH-001 growth phase: in mid-logarithmic phase stress: control
|
Treatment protocol |
Cells were collected and shifted to the fresh chemical medium at pH 7.0 (control cells) and 4.0 (acid-stressed cells), respectively. All cultures were incubated at 30 °C for 2 h prior to RNA extraction.
|
Growth protocol |
The mixture of K. vulgare and B. megaterium was grown at 30 °C in the well defined chemical medium at pH 7.0 for 18 h until cells were at their mid-log phase.
|
Extracted molecule |
total RNA |
Extraction protocol |
In order to stabilize the bacterial transcriptome profile, one-sixth of the culture volume of ice-cold 5% phenol-ethanol stop solution (v/v) was added to the culture firstly. After stabilizing the cells, the extraction of total RNA was conducted using the RNeasy Bacteria Mini kit (Qiagen) according to the manufacturer’s protocol. During the RNA extraction process, trace elements of remaining DNA were eliminated by DNase digestion using the RNase-Free DNase Set (Qiagen). The concentration and purity of the total RNA were assessed by Nanodrop® ND-8000 (Thermo Fisher), and the integrity of the RNA was assessed using standard denaturing agarose gel electrophoresis.
|
Label |
Cy5
|
Label protocol |
Sample labeling and hybridization reactions were performed according to the Agilent Two-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, for each sample 1 μg of total RNA was linearly amplified and labeled with Cy5-dCTP and sample of common reference with Cy3-dCTP. Labelled cRNAs purified with Qiagen RNAeasy Mini Kit, and sample concentration and specific activity (pmol Cy5/Cy3 per μg cRNA) were measured in a NanoDrop® ND-8000 spectrophotometer.
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Channel 2 |
Source name |
pooled 3 control and 3 acid-stressed cells
|
Organism |
Ketogulonicigenium vulgare |
Characteristics |
reference: pooled 3 control and 3 acid-stressed cells strain: WSH-001 growth phase: in mid-logarithmic phase
|
Treatment protocol |
Cells were collected and shifted to the fresh chemical medium at pH 7.0 (control cells) and 4.0 (acid-stressed cells), respectively. All cultures were incubated at 30 °C for 2 h prior to RNA extraction.
|
Growth protocol |
The mixture of K. vulgare and B. megaterium was grown at 30 °C in the well defined chemical medium at pH 7.0 for 18 h until cells were at their mid-log phase.
|
Extracted molecule |
total RNA |
Extraction protocol |
In order to stabilize the bacterial transcriptome profile, one-sixth of the culture volume of ice-cold 5% phenol-ethanol stop solution (v/v) was added to the culture firstly. After stabilizing the cells, the extraction of total RNA was conducted using the RNeasy Bacteria Mini kit (Qiagen) according to the manufacturer’s protocol. During the RNA extraction process, trace elements of remaining DNA were eliminated by DNase digestion using the RNase-Free DNase Set (Qiagen). The concentration and purity of the total RNA were assessed by Nanodrop® ND-8000 (Thermo Fisher), and the integrity of the RNA was assessed using standard denaturing agarose gel electrophoresis.
|
Label |
Cy3
|
Label protocol |
Sample labeling and hybridization reactions were performed according to the Agilent Two-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, for each sample 1 μg of total RNA was linearly amplified and labeled with Cy5-dCTP and sample of common reference with Cy3-dCTP. Labelled cRNAs purified with Qiagen RNAeasy Mini Kit, and sample concentration and specific activity (pmol Cy5/Cy3 per μg cRNA) were measured in a NanoDrop® ND-8000 spectrophotometer.
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|
|
Hybridization protocol |
A total of 1μg of Cy3-labeled and Cy5-labeled cRNA was prepared for fragmentation adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, heated at 60 °C for 30 min, and finally diluted by addition with 55μl 2×GE Hybridization buffer. A volume of 100 μl of hybridization solution was then dispensed in the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven.
|
Scan protocol |
The hybridized arrays were washed, fixed and scanned using the Agilent DNA microarray scanner (part number G2505B) following Agilent Technologies’ guidelines.
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Data processing |
Agilent Feature Extraction Software (version 10.5.1.1) was used to analyze acquired array images. Lowess normalization and subsequent data processing was performed using the Agilent GeneSpring GX v11.5.1 software. Following Lowess normalization of the raw data, genes that at least 8 out of 16 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance were identified through Volcano Plot filtering. The threshold we used to screen up- or downregulated genes was defined by Fold Change ≥2 and P-value <0.05.
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Submission date |
Oct 20, 2011 |
Last update date |
Oct 23, 2014 |
Contact name |
Haoru Yang |
E-mail(s) |
haoruyang@gmail.com
|
Organization name |
Jiangnan University
|
Street address |
1800 Lihu Road
|
City |
Wuxi |
ZIP/Postal code |
214122 |
Country |
China |
|
|
Platform ID |
GPL14693 |
Series (1) |
GSE33105 |
Ketogulonicigenium vulgare cells: Acid-stressed vs. Control |
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