|
| Status |
Public on Jul 24, 2010 |
| Title |
Roseburia Inulinivorans_inulin3_starch5_A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mRNA extracted from Roseburia Inulinivorans grown on Inulin at OD 0.4
|
| Organism |
Roseburia inulinivorans DSM 16841 |
| Characteristics |
strain: A2-194
|
| Growth protocol |
Routine anaerobic culturing of R. inulinivorans A2-194 was in M2GSC medium (39). Growth on single carbon sources utilised basal YCFA medium (3) supplemented with 0.5 % w/v of the specific substrate (inulin -Dahlia, Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified from mid-exponential phase (OD650 = 0.4) cultures on basal YCFA supplemented with inulin using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion).
|
| Label |
Cy3
|
| Label protocol |
The purified RNA (1 ug) was labelled by reverse transcription (Amersham Cyscribe first-strand cDNA labelling kit), employing random nonamer extension incorporating either dCTP-Cy3 dye.
|
| |
|
| Channel 2 |
| Source name |
mRNA extracted from Roseburia Inulinivorans grown on starch at OD 0.4
|
| Organism |
Roseburia inulinivorans DSM 16841 |
| Characteristics |
strain: A2-194
|
| Growth protocol |
Routine anaerobic culturing of R. inulinivorans A2-194 was in M2GSC medium (39). Growth on single carbon sources utilised basal YCFA medium (3) supplemented with 0.5 % w/v of the specific substrate (amylopectin starch, Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified from mid-exponential phase (OD650 = 0.4) cultures on basal YCFA supplemented with starch using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion).
|
| Label |
Cy5
|
| Label protocol |
The purified RNA (1 ug) was labelled by reverse transcription (Amersham Cyscribe first-strand cDNA labelling kit), employing random nonamer extension incorporating dCTP-Cy5 dye.
|
| |
|
| |
| Hybridization protocol |
Hybridization was done in the GeneTAC hybridization station (Genomic Solutions) with agitation and using circulating buffers. Slides were first blocked in buffer (180mM succinic anhydride, 44mM sodium borate in 1-methyl-2-pyrrolidone), washed in deionised water at 98°C (2× 1min) and finally in 95 % ethanol (1 min) at room temperature. After drying the slides were pre-hybridized at 50°C for 30 min (in 10 mg ml17 fraction V BSA, 3.5× SSC and 0.1% SDS), washed again in deionised water, dried and immediately hybridized. The hybridization solution (labelled cDNA mixed with 10µg Human Cot-1 DNA (Invitrogen), 15.4 µg yeast tRNA, 8 µl poly dA, 14.4 µl 20× SSC and 2.4 µl 100× Denhardt’s solution) was first boiled for 2 min and cooled to 45°C before adding 1.4 µl 5% SDS and incubating at 65°C for 30 min. This solution was then added to the array slides which were hybridized for 16 h at 65°C before washing at room temperature in medium stringency buffer (0.1 % SDS, 0.5× SSC) for 5 min, followed by two high stringency washes (0.1 % SDS, 0.1× SSC) of 5 min each.
|
| Scan protocol |
The fluorescence of each spot was measured in two channels using a GeneTAC LS IV (Genomic Solutions) utilising GeneTac Integrator version 3.0.1 software.
|
| Description |
Strain Roseburia inulinivorans A2-194 was isolated from a human faecal sample in 1997, and is a member of the Firmicutes
|
| Data processing |
The microarray data were log2-transformed and normalised by Loess normalisation to remove intensity dependent dye-effects. As each clone was represented by three spots we calculated the median of the three log2(Cy5/Cy3) values, a robust measure of relative gene expression. In total the fluorescence of 12 hybridising spots were compared for each clone. P-values were calculated by applying a one-sample t-test to the two log-ratios from the two replicate experiments, while the two dye-swapped arrays within each experiment were combined by averaging.
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| |
|
| Submission date |
Jun 09, 2010 |
| Last update date |
Jul 23, 2010 |
| Contact name |
Gill Campbell |
| E-mail(s) |
[email protected]
|
| Phone |
+44 (0) 1224 716627
|
| Fax |
+44 (0) 1224 716647
|
| URL |
http://www.rowett.ac.uk
|
| Organization name |
University of Aberdeen
|
| Department |
Rowett Institute of Nutrition and Health
|
| Street address |
Greenburn Road
|
| City |
Aberdeen |
| ZIP/Postal code |
AB21 9SB |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10020 |
| Series (1) |
| GSE22245 |
Differential gene expression in Roseburia inulinivorans grown on inulin and starch |
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