|
| Status |
Public on Oct 22, 2009 |
| Title |
LB broth with 0.5% glucose (OD1.0)[rep1_1_a] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mutant at OD1.0
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
PJ002 luxS mutant grown in LB broth with 0.5% glucose
|
| Biomaterial provider |
mutant created by Palmy Jesudhasan
|
| Treatment protocol |
mutant cells were grown in LB broth with 0.5% glucose until it reaches OD 1.0
|
| Growth protocol |
Grown at 37º C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the cultures using an RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. RNAprotect bacteria reagent (QIAGEN, Inc., Valencia, CA) was added to the cultures to stabilize RNA before isolation. The RNase-free DNase set (QIAGEN, Inc., Valencia, CA) was used for on-column DNase digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE) and bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) respectively.
|
| Label |
Cy5
|
| Label protocol |
Total RNA (10 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNAs from the mutant was labeled with Cy-5 mono-Reactive Dye (GE Health Care).
|
| |
|
| Channel 2 |
| Source name |
wild type at OD1.0
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
Salmonella Typhimurium grown in LB with 0.5% glucose
|
| Biomaterial provider |
National Veterinary Service Laboratory, Ames, Iowa
|
| Treatment protocol |
wild type cells were grown in LB broth with 0.5% glucose until it reaches OD 1.0
|
| Growth protocol |
Grown at 37º C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the cultures using an RNeasy mini kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. RNAprotect bacteria reagent (QIAGEN, Inc., Valencia, CA) was added to the cultures to stabilize RNA before isolation. The RNase-free DNase set (QIAGEN, Inc., Valencia, CA) was used for on-column DNase digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE) and bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) respectively.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (10 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNAs from the wild type was labeled with Cy-5 mono-Reactive Dye (GE Health Care).
|
| |
|
| |
| Hybridization protocol |
The labeling mixtures were further purified using a QIAquick PCR purification kit (Qiagen). Equal amounts of labeled cDNA from the treatment and control were used to hybridize S. Typhimurium LT2 genome microarrays (version 4), obtained from The Institute for Genomic Research (TIGR, Rockville, MD). These arrays were 70 mer-oligo arrays with 4504 ORF each, with 5 replicate spots per ORF. The labeled cDNA was applied to the above arrays. Hybridization was carried out overnight at 42°C in a water bath using Corning hybridization chamber. The pre-and post-hybridization was carried out using the the TIGR SOP #M008.
|
| Scan protocol |
The slides was washed and scanned using a GenePix 4100A scanner (Axon Instruments Inc., Union City, CA) at 532 nm (Cy3 channel) and 635 nm (Cy5 channel), and the image was stored for further analysis.
|
| Description |
The slide was prehybridized following the TIGR SOP # M007
|
| Data processing |
The data was normalized in Acuity 4.0 using Lowess normalization method
|
| |
|
| Submission date |
Apr 17, 2007 |
| Last update date |
Oct 22, 2009 |
| Contact name |
Palmy R Jesudhasan |
| E-mail(s) |
palmy.jesudhasan@utsouthwestern.edu
|
| Phone |
214-648-5627
|
| Organization name |
University of Texas Southwestern Medical Center
|
| Department |
Microbiology
|
| Street address |
NL4.110M
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
| |
|
| Platform ID |
GPL5096 |
| Series (1) |
| GSE7558 |
Salmonella enterica Typhimurium grown with and without glucose |
|
