|
| Status |
Public on Oct 01, 2011 |
| Title |
LuxS_vs._WT_Dilute_medium_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
luxS mutant Lactobacillus reuteri 100-23 grown in dlute MRS
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth stage: Exponential
strain: LuxS mutant
|
| Treatment protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium for four hours, eight hours or under dilute medium culture on reaching an OD600 of 0.4.
|
| Growth protocol |
Lactobacillus reuteri 100-23 was cultured in Lactobacilli MRS medium (Difco, Becton Dickinson Co., Sparks, MD, USA) incubated anaerobically at 37oC. Lactobacilli MRS medium was either full strength of diluted to one-fifth concentration according to experimental requirements. Liquid media were filter-sterilised, agar media were autoclaved.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cells were harvested by brief centrifugation at 12,000 x g and washed with 750 µl of RNAprotect reagent (Qiagen). The bacterial cells were disrupted by bead beating (5000 rpm, 2 x 40 seconds) in TRIzol™ reagent (Invitrogen) and extracted with chloroform. RNA was precipitated with iso-propanol and dried after removal of iso-propanol. The dried RNA was then dissolved in nuclease-free water. The RNA was further purified using the RNeasy Mini Kit (Qiagen) and DNase treated using a DNA-free kit (Ambion). RNA quality was assessed using a Bioanalyzer 2100 instrument (Agilent Technologies) and quantified by NanoDrop ND-1000 (Nanodrop).
|
| Label |
Cy3
|
| Label protocol |
Two micrograms of total RNA was labelled with either Cy5- or Cy3-dCTP during first-strand synthesis with SuperScript II (Invitrogen) reverse transcriptase kit following the manufacturer’s protocol. Labelled cDNA was purified using a MinElute PCR purification kit (Qiagen) and the dye incorporation rate was determined by NanoDrop ND-1000 UV-Vis spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Wild type Lactobacillus reuteri 100-23 grown in dlute MRS
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth stage: Exponential
strain: Wild type
|
| Treatment protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium for four hours, eight hours or under dilute medium culture on reaching an OD600 of 0.4.
|
| Growth protocol |
Lactobacillus reuteri 100-23 was cultured in Lactobacilli MRS medium (Difco, Becton Dickinson Co., Sparks, MD, USA) incubated anaerobically at 37oC. Lactobacilli MRS medium was either full strength of diluted to one-fifth concentration according to experimental requirements. Liquid media were filter-sterilised, agar media were autoclaved.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cells were harvested by brief centrifugation at 12,000 x g and washed with 750 µl of RNAprotect reagent (Qiagen). The bacterial cells were disrupted by bead beating (5000 rpm, 2 x 40 seconds) in TRIzol™ reagent (Invitrogen) and extracted with chloroform. RNA was precipitated with iso-propanol and dried after removal of iso-propanol. The dried RNA was then dissolved in nuclease-free water. The RNA was further purified using the RNeasy Mini Kit (Qiagen) and DNase treated using a DNA-free kit (Ambion). RNA quality was assessed using a Bioanalyzer 2100 instrument (Agilent Technologies) and quantified by NanoDrop ND-1000 (Nanodrop).
|
| Label |
Cy5
|
| Label protocol |
Two micrograms of total RNA was labelled with either Cy5- or Cy3-dCTP during first-strand synthesis with SuperScript II (Invitrogen) reverse transcriptase kit following the manufacturer’s protocol. Labelled cDNA was purified using a MinElute PCR purification kit (Qiagen) and the dye incorporation rate was determined by NanoDrop ND-1000 UV-Vis spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
The labelled samples were mixed and competitively hybridised using an Agilent Gene Expression Hybridisation Kit (part number: 5188-5242) in an Agilent hybridization oven (G2545A) at 65°C for 24 hours.
|
| Scan protocol |
Microarray slides were scanned using a GenePix 400B Scanner (Axon Instruments, USA) and the GenePix Pro 6.0 software. The slides were scanned at 5 μm and a photo-multiplier tube (PMT) setting of 720 for the red channel (635 nm laser measuring the CyTM5 labelled nucleic acid) and 630 for the green channel (532 nm laser measuring the CyTM3 labelled nucleic acid). The GenePix array list (GAL) file was used for extraction of the microarray results.
|
| Description |
Wild type
|
| Data processing |
Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5.3.1) which performs a lowess normalization. Microarray data was processed as described by García de la Nava et al 2003 and van Hijum et al 2005. Differential gene expression was determined by Cyber-T test (Long 2001).
|
| |
|
| Submission date |
Sep 30, 2011 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Charlotte Wilson |
| Organization name |
ORNL
|
| Department |
Biosciences Bldg 1520
|
| Lab |
329
|
| Street address |
1 Bethel Valley Road
|
| City |
Oak Ridge |
| State/province |
Tennessee |
| ZIP/Postal code |
37831-6342 |
| Country |
New Zealand |
| |
|
| Platform ID |
GPL10986 |
| Series (1) |
| GSE32520 |
Transcriptional and metabolomic consequences of luxS inactivation reveal a metabolic rather than quorum sensing role for LuxS in Lactobacillus reuteri 100-23 |
|
