|
| Status |
Public on Jul 31, 2015 |
| Title |
AI-2 treated Cy3 vs. untreated Cy5 rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Salmonella Typhimurium
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
sample type: pj002 (luxs: :cat), luxS mutant of this strain of S. Typhimurium (Accession # 87-26254) poultry isolate obtained from the National Veterinary Service Laboratory, Ames, Iowa.
treatment: 10% PBS (control)
genotype: luxS mutant
|
| Biomaterial provider |
PFGRC
|
| Treatment protocol |
Salmonella Typhimurium with 10% PBS (control).
|
| Growth protocol |
Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% PBS for 3 hours.
|
| 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 (5 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 cDNA was labeled with Cy-5 mono-Reactive Dye (GE Health Care).
|
| |
|
| Channel 2 |
| Source name |
Salmonella Typhimurium
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
sample type: pj002 (luxs: :cat), luxS mutant of this strain of S. Typhimurium (Accession # 87-26254) poultry isolate obtained from the National Veterinary Service Laboratory, Ames, Iowa.
treatment: 25 mM in vitro synthesized AI-2 (Treatment)
genotype: luxS mutant
|
| Biomaterial provider |
PFGRC
|
| Treatment protocol |
Salmonella Typhimurium treated with 25 mM in vitro synthesized AI-2 (Treatment).
|
| Growth protocol |
Fresh Salmonella Typhimurium was allowed to grow in the presence of 10% (25 mM) in vitro synthesized AI-2 for 3 hours.
|
| 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 (5 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 cDNA was labeled with Cy-3 mono-Reactive Dye (GE Health Care).
|
| |
|
| |
| Hybridization protocol |
The labeled 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 on S. Typhimurium LT2 genome microarrays (version 2), obtained from JCVI (formerly, TIGR, Rockville, MD) and provided by the Pathogen Functional Genome Resource Center (PFGRC). These arrays were amplicon arrays with 6780 ORF each, with 2 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 PFGRC SOP #M008.
|
| Scan protocol |
The slides were 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 images were stored for further analysis.
|
| Description |
The slides were prehybridized following PFGRC's SOP # M007.
|
| Data processing |
The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. The normalized data was analyzed using commercial SAS 9.1.3 program (SAS Institute Inc. Cary, NC).
|
| |
|
| Submission date |
Aug 12, 2010 |
| Last update date |
Jul 31, 2015 |
| 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 |
GPL10785 |
| Series (2) |
| GSE23588 |
Influence of Autoinducer-2 (AI-2) in Salmonella Typhimurium Gene Expression |
| GSE23761 |
Influence of Autoinducer-2 (AI-2) in Cell-Free Supernatant of Salmonella Typhimurium in Gene Expression of S. Typhimurium |
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