|
| Status |
Public on Sep 30, 2012 |
| Title |
in vivo vs. in vitro log phase, rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lactobacillus reuteri 100-23 grown in MRS to log phase
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth type: in vitro
growth phase: log
|
| Growth protocol |
In vitro: Lactobacillus reuteri 100-23 was cultured in Lactobacilli MRS medium (Difco, Becton Dickinson Co., Sparks, MD, USA) incubated anaerobically at 37oC. Liquid media were filter-sterilised, and agar media were autoclaved.
In vivo: Groups of six to eight female Lactobacillus-free BALB/C mice were anaesthetised using 250 mg/kg body weight avertin injected intraperitoneally prior to inoculation with 100 µl of L. reuteri 100-23C suspended in 0.9% (w/v) NaCl by intragastric gavage (109 cells per ml). After two weeks, the animals were sacrificed by cervical dislocation and the stomach contents and scrapings of the stomach surface used for RNA extraction. For the control hybridisation, Lactobacillus-free BALB/C mice were sacrificed by cervical dislocation and the stomach contents and scrapings of the stomach surface used for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium for four hours and eight hours and from cells harvested from the stomachs of mice colonised for 14_1 days with L. reuteri 100-23.
From bacterial culture (in vitro): 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 4_10 seconds) in TRIzol reagent (Invitrogen) and extracted with chloroform. RNA was precipitated with isopropanol and dried after removal of isopropanol. 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).
From the mouse stomach (in vivo): Stomach contents and scrapings from the forestomach surface of mice were washed once with RNAprotect Bacteria Reagent. Samples were centrifuged at 150 x g for 5 min at 5°C, and the supernatant was further centrifuged at 300 x g for 5 min at 5°C to remove debris. Cells were recovered by centrifugation at 5000 x g for 5 min at 5°C. Cell pellets from 6-8 mice were pooled in 1 ml of TRIzol Reagent and cells were disrupted by bead beating (5000 rpm, 2 x 4_10 seconds) and extracted with chloroform. RNA was precipitated with isopropanol and dried after removal of isopropanol. 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). Contaminating mouse RNA from the stomach-derived samples of L. reuteri 100-23 RNA was removed with the MICROBEnrich Kit (Ambion, USA).
|
| Label |
Cy3
|
| Label protocol |
Five hundred nanograms of total RNA was used as a template for RNA amplification using the MessageAmp II Bacteria kit (Ambion, USA) according to the manufacturer's protocol. During the in vitro transcription step of the RNA amplification protocol to synthesize amplified RNA (aRNA), the modified nucleotide, 5-(3-aminoallyl)-UTP (aa-UTP) (Ambion, USA), was incorporated into the aRNA in equal proportion to unmodified UTP. Ten micrograms of purified aRNA was labelled with either Cy3 or Cy5 Post-labelling Reactive Dyes (Amersham biosciences, USA). The labelled aRNA was purified using the RNeasy Kit (Qiagen) following the RNA cleanup protocol, and the dye incorporation rate was determined by a NanoDrop ND-1000 UV-Vis spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Lactobacillus reuteri 100-23 harvested from the mouse stomach, two weeks after intragastric inoculation of reconstituted-Lactobacillus free mice
|
| Organism |
Limosilactobacillus reuteri subsp. rodentium |
| Characteristics |
growth type: in vivo
growth source: stomach of inoculated mouse
|
| Growth protocol |
In vitro: Lactobacillus reuteri 100-23 was cultured in Lactobacilli MRS medium (Difco, Becton Dickinson Co., Sparks, MD, USA) incubated anaerobically at 37oC. Liquid media were filter-sterilised, and agar media were autoclaved.
In vivo: Groups of six to eight female Lactobacillus-free BALB/C mice were anaesthetised using 250 mg/kg body weight avertin injected intraperitoneally prior to inoculation with 100 µl of L. reuteri 100-23C suspended in 0.9% (w/v) NaCl by intragastric gavage (109 cells per ml). After two weeks, the animals were sacrificed by cervical dislocation and the stomach contents and scrapings of the stomach surface used for RNA extraction. For the control hybridisation, Lactobacillus-free BALB/C mice were sacrificed by cervical dislocation and the stomach contents and scrapings of the stomach surface used for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultures of strain 100-23 grown in Lactobacilli MRS medium for four hours and eight hours and from cells harvested from the stomachs of mice colonised for 14_1 days with L. reuteri 100-23.
From bacterial culture (in vitro): 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 4_10 seconds) in TRIzol reagent (Invitrogen) and extracted with chloroform. RNA was precipitated with isopropanol and dried after removal of isopropanol. 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).
From the mouse stomach (in vivo): Stomach contents and scrapings from the forestomach surface of mice were washed once with RNAprotect Bacteria Reagent. Samples were centrifuged at 150 x g for 5 min at 5°C, and the supernatant was further centrifuged at 300 x g for 5 min at 5°C to remove debris. Cells were recovered by centrifugation at 5000 x g for 5 min at 5°C. Cell pellets from 6-8 mice were pooled in 1 ml of TRIzol Reagent and cells were disrupted by bead beating (5000 rpm, 2 x 4_10 seconds) and extracted with chloroform. RNA was precipitated with isopropanol and dried after removal of isopropanol. 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). Contaminating mouse RNA from the stomach-derived samples of L. reuteri 100-23 RNA was removed with the MICROBEnrich Kit (Ambion, USA).
|
| Label |
Cy5
|
| Label protocol |
Five hundred nanograms of total RNA was used as a template for RNA amplification using the MessageAmp II Bacteria kit (Ambion, USA) according to the manufacturer's protocol. During the in vitro transcription step of the RNA amplification protocol to synthesize amplified RNA (aRNA), the modified nucleotide, 5-(3-aminoallyl)-UTP (aa-UTP) (Ambion, USA), was incorporated into the aRNA in equal proportion to unmodified UTP. Ten micrograms of purified aRNA was labelled with either Cy3 or Cy5 Post-labelling Reactive Dyes (Amersham biosciences, USA). The labelled aRNA was purified using the RNeasy Kit (Qiagen) following the RNA cleanup protocol, and the dye incorporation rate was determined by a NanoDrop ND-1000 UV-Vis spectrophotometer.
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| |
|
| |
| Hybridization protocol |
The labelled samples were mixed and competitively hybridised using an Agilent Gene Expression Hybridisation Kit (part number: 5188-524_12) in an Agilent hybridization oven (G254_15A) at 65°C for 24_1 hours.
|
| Scan protocol |
In vivo vs. in vitro arrays: Microarray slides were scanned using a GenePix 4_100B Scanner (Axon Instruments, USA) and the GenePix Pro 6.0 software. The slides were scanned at 5 μm and a photomultiplier tube (PMT) setting of 780 for the red channel (635 nm laser measuring the Cy5 labelled nucleic acid) and 510 for the green channel (532 nm laser measuring the Cy3 labelled nucleic acid). The GenePix array list (GAL) file was used for extraction of the microarray results.
Control hybridisation: 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 9.5.3.1) 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).
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| Submission date |
Mar 05, 2012 |
| Last update date |
Sep 30, 2012 |
| 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) |
| GSE36286 |
Inactivation of the Lactobacillus reuteri 100-23 ureC gene affects in vitro acid tolerance, and impairs ecological fitness in vivo |
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