|
| Status |
Public on Dec 11, 2010 |
| Title |
Maltose_vs_Glucose_dyeswap_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lactobacillus reuteri 100-23 grown in the presence of glucose
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth stage: exponential
carbon source: glucose
|
| Treatment protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium containing 2% (w/v) glucose or maltose for four hours.
|
| 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 made from scratch and contained either glucose or maltose at 2% (w/v) 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 |
Lactobacillus reuteri 100-23 grown in mMRS with maltose
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth stage: exponential
carbon source: maltose
|
| Treatment protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium containing 2% (w/v) glucose or maltose for four hours.
|
| 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 made from scratch and contained either glucose or maltose at 2% (w/v) 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 |
Microarrays were scanned using the Agilent Microarray Scanner System (G2505B) and Agilent scan control software version 7.0 at a resolution of 5 micrometres
|
| Data processing |
Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.1) 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). Genes with a P value <0.001 and greater than two fold difference under the two conditions were considered differentially expressed.
|
| |
|
| Submission date |
Sep 30, 2010 |
| Last update date |
Dec 11, 2010 |
| 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) |
| GSE24472 |
Maltose utilization by Lactobacillus reuteri 100-23 |
|
