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Sample GSM819917 Query DataSets for GSM819917
Status Public on Oct 23, 2014
Title Acid-Stressed K.vulgare Cell Replicate 2
Sample type RNA
 
Channel 1
Source name Acid-stressed K.vulgare cells
Organism Ketogulonicigenium vulgare
Characteristics strain: WSH-001
growth phase: in mid-logarithmic phase
stress: pH 4.0 treated for 2 h
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.
 
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.
 
 
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.
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.
 
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

Data table header descriptions
ID_REF
VALUE Lowess normalized log2 ratio (test/reference) data

Data table
ID_REF VALUE
KVU_1159 -0.11697674
KVU_1530 -0.4161806
KVU_2095 -0.43597794
KVU_2223 -0.052437782
KVU_PA0140 0.086502075
KVU_2209 -0.43955898
KVU_1130 0.1521635
KVU_1202 0.61658573
KVU_0781 -0.607193
KVU_2094 -0.4469738
KVU_PA0060 0.09346485
KVU_2322 -0.10406399
KVU_1973 0.076545715
KVU_1525 0.48384762
KVU_0467 -0.101192474
KVU_1527 -0.4595585
KVU_0536 -0.21838093
KVU_0627 -0.6074829
KVU_PA0018 0.30223465
KVU_1626 0.007158279

Total number of rows: 2301

Table truncated, full table size 46 Kbytes.




Supplementary file Size Download File type/resource
GSM819917_Acid-stressed-rep2.txt.gz 1.4 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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