|
Status |
Public on Apr 26, 2012 |
Title |
Co-cultured K. vulgare Cells, Replicate 3 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Co-cultured K. vulgare cells
|
Organisms |
Priestia megaterium; Ketogulonicigenium vulgare WSH-001 |
Characteristics |
growth condition: co-culture with Bacillus megaterium growth phase: mid-logarithmic
|
Treatment protocol |
The mixture of K. vulgare and B. megaterium was grown at 30 °C in the well-defined chemical medium at pH 7.0, 200 rpm for 18 h until cells were at their mid-log phase.
|
Growth protocol |
The mono-culture of K. vulgare was grown at 30 °C in the well-defined chemical medium at pH 7.0, 200rpm for 18 h.
|
Extracted molecule |
total RNA |
Extraction protocol |
The mono- and co-cultures were sampled after 18-h fermentation (mid-log phase) and added to RNAProtect® Bacterial Reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Bacterial cells were treated with lysozyme stock solution (40 mg ml-1 in 30 mM pH 8.0 Tris/EDTA buffer) and protease K at 25oC for 10 min (vortex 10 s for every 2 min). Total RNA was isolated and purified using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Total RNA binding on the RNeasy spin column membrane were treated with RNase-Free DNase Set (Qiagen) to remove remaining DNA. 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 were purified with the Qiagen RNeasy Mini Kit, and sample concentration and specific activity (pmol Cy5/Cy3 per μg cRNA) were measured in a NanoDrop® ND-8000 spectrophotometer.
|
|
|
Channel 2 |
Source name |
Pooled 3 co-cultured and 3 mono-cultured cells
|
Organisms |
Priestia megaterium; Ketogulonicigenium vulgare WSH-001 |
Characteristics |
growth condition: co-culture with Bacillus megaterium and mono-culture growth phase: mid-logarithmic sample type: reference
|
Treatment protocol |
The mixture of K. vulgare and B. megaterium was grown at 30 °C in the well-defined chemical medium at pH 7.0, 200 rpm for 18 h until cells were at their mid-log phase.
|
Growth protocol |
The mono-culture of K. vulgare was grown at 30 °C in the well-defined chemical medium at pH 7.0, 200rpm for 18 h.
|
Extracted molecule |
total RNA |
Extraction protocol |
The mono- and co-cultures were sampled after 18-h fermentation (mid-log phase) and added to RNAProtect® Bacterial Reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Bacterial cells were treated with lysozyme stock solution (40 mg ml-1 in 30 mM pH 8.0 Tris/EDTA buffer) and protease K at 25oC for 10 min (vortex 10 s for every 2 min). Total RNA was isolated and purified using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Total RNA binding on the RNeasy spin column membrane were treated with RNase-Free DNase Set (Qiagen) to remove remaining DNA. 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 were purified with the Qiagen RNeasy Mini Kit, and sample concentration and specific activity (pmol Cy5/Cy3 per μg cRNA) were measured in a NanoDrop® ND-8000 spectrophotometer.
|
|
|
|
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 of 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 Technology’s guidelines.
|
Description |
Co-cultured-rep3 Common reference design.
|
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 for which 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.
|
|
|
Submission date |
Apr 25, 2012 |
Last update date |
Apr 26, 2012 |
Contact name |
Zhu Yibo |
E-mail(s) |
centuryrain@126.com
|
Organization name |
Jiangnan University
|
Street address |
Lihu Road 1800#
|
City |
Wuxi |
State/province |
Jiangsu |
ZIP/Postal code |
214122 |
Country |
China |
|
|
Platform ID |
GPL14693 |
Series (1) |
GSE37594 |
Ketogulonigenium vulgare cells: co-culture mode vs. mono-culture mode |
|