NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM238758 Query DataSets for GSM238758
Status Public on Nov 14, 2007
Title SmutansUA159_Mupricin_rep4
Sample type RNA
 
Channel 1
Source name S. mutans UA159 mup treated
Organism Streptococcus mutans
Characteristics Strain UA159
Biomaterial provider Dr. Marcelle Nascimento
Treatment protocol Treated with 500 ng/ml of mupirocin for 20 minutes at 37°C once OD600 of 0.3 was reached
Growth protocol Cells were grown in the chemically-defined medium FMC to an OD600 of 0.3 in 5% CO2 at 37°C
Extracted molecule total RNA
Extraction protocol RNA from S. mutans was isolated from 50 ml cultures that were grown under the desired conditions and harvested by centrifugation at 4ºC. Pelleted cells were resuspended in 400 ml of DEPC-treated water, 800 ml of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at one minute intervals. Cells were then pelleted, resuspended in 500 ml Tris-EDTA (50:10) buffer and transferred to 1.5 ml screw cap tubes containing sterile glass beads (0.1 mm avg. diam.; Biospec, Bartlesville, OK), 100 ml of 1% SDS and 650 ml of acid phenol:chloroform (5:1). The mixture was homogenized in a Bead Beater twice for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4ºC. Two additional hot acid phenol:chloroform extractions were performed followed by one extraction with chloroform:isoamyl-alcohol (24:1). RNA was precipitated in the presence of isopropanol and sodium acetate, pH 5, at -20ºC for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in DEPC-treated water and digested with DNAseI (Ambion, Austin, TX). The RNA was re-purified and treated on-column with DNaseI using the RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA), as recommended by the supplier.
Label Cy3
Label protocol All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy3)-dUTP
 
Channel 2
Source name S. mutans UA159 grown in BHI
Organism Streptococcus mutans
Characteristics Strain UA159
Biomaterial provider Dr. Marcelle Nascimento
Growth protocol Growth Media: Brain Heart Infusion
OD600: 0.5
5% CO2, 37°C
Extracted molecule total RNA
Extraction protocol RNA from S. mutans was isolated from 50 ml cultures that were grown under the desired conditions and harvested by centrifugation at 4ºC. Pelleted cells were resuspended in 400 ml of DEPC-treated water, 800 ml of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at one minute intervals. Cells were then pelleted, resuspended in 500 ml Tris-EDTA (50:10) buffer and transferred to 1.5 ml screw cap tubes containing sterile glass beads (0.1 mm avg. diam.; Biospec, Bartlesville, OK), 100 ml of 1% SDS and 650 ml of acid phenol:chloroform (5:1). The mixture was homogenized in a Bead Beater twice for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4ºC. Two additional hot acid phenol:chloroform extractions were performed followed by one extraction with chloroform:isoamyl-alcohol (24:1). RNA was precipitated in the presence of isopropanol and sodium acetate, pH 5, at -20ºC for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in DEPC-treated water and digested with DNAseI (Ambion, Austin, TX). The RNA was re-purified and treated on-column with DNaseI using the RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA), as recommended by the supplier.
Label Cy5
Label protocol All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy5)-dUTP
 
 
Hybridization protocol Hybridizations were carried out in a Maui 4-chamber hybridization system (BioMicro Systems, Salt Lake City, Utah) for 17 h at 42ºC. The slides were then washed according to TIGR protocols (SOT# M008, version 2.1, 11/06).
Scan protocol The slides were scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA).
Description Streptococcus mutans wildtype, mupirocin treated
Data processing After the slides were scanned, single-channel images were loaded into TIGR Spotfinder software and overlayed. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, then the intensity values of each spot were measured. Data was normalized using Microarray data analysis software (MIDAS), by using LOWESS and Iterative Log Mean Centering with default settings.
 
Submission date Oct 19, 2007
Last update date Aug 14, 2011
Contact name Robert A Burne
E-mail(s) [email protected]
Phone 352-846-2520
Organization name University of Florida
Department Oral Biology
Lab Burne Lab
Street address 1600 SW Archer Rd. BOX 100424
City Gainesville
State/province FL
ZIP/Postal code 32610-0424
Country USA
 
Platform ID GPL4340
Series (1)
GSE9382 The Role of RelA of Streptococcus mutans in Global Control of Gene Expression

Data table header descriptions
ID_REF
VALUE Log2 of the Channel A/Channel B ratio

Data table
ID_REF VALUE
1 -0.78
2 -1.85
3 -1.73
4 -0.47
5 -2.23
6 0.28
8 -1.86
9 -0.87
10 -0.76
11 -1.22
12 -1.6
13 -0.34
14 -1.44
15 -1.39
16 0.42
17 -0.83
18 -0.51
19 -1.29
20 -1.02
21 0.89

Total number of rows: 7557

Table truncated, full table size 75 Kbytes.




Supplementary file Size Download File type/resource
GSM238758.mev.gz 233.3 Kb (ftp)(http) MEV
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap