|
| Status |
Public on Jan 01, 2016 |
| Title |
PRL2010 cultivated on kefiran as unique carbon source II |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PRL2010_MRS plus kefiran_12h
|
| Organism |
Bifidobacterium bifidum |
| Characteristics |
strain: PRL2010
cultivated on: MRS plus kefiran for 12h
|
| Growth protocol |
B. bifidum PRL2010 was grown anaerobically in the Man-Rogosa-Sharp (MRS) with 2% of kefiran supplemented with 0.05% (w/v) L-cysteine hydrochloride and incubated at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, cell pellets/tissue materials were resuspended in 1 ml of QUIAZOL (Qiagen, UK) and placed in a tube containing 0.8 g of glass beads (diameter, 106 μm; Sigma). The cells were lysed by shaking the mix on a BioSpec homogenizer at 4°C for 2 min (maximum setting). The mixture was then centrifuged at 12,000 rpm for 15 min, and the upper phase containing the RNA-containing sample was recovered. The RNA sample was further purified by phenol extraction and ethanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription and amplification of 500 ng of total RNA was performed with ImProm-IITM Reverse Transcriptase (Promega, Madison, USA) according to the manufacturer’s instructions. 5 µg of RNA was then labelled with ULS Labelling kit with Cy5 or Cy3 (Kreatech, The Netherlands). For the purpose of the tiling array, samples containing kefir/kefiran cDNA was labelled with Cy5, while control samples (MRS) were labelled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
PRL2010_MRS (control)
|
| Organism |
Bifidobacterium bifidum |
| Characteristics |
strain: PRL2010
cultivated on: MRS (control)
|
| Growth protocol |
B. bifidum PRL2010 was grown anaerobically in the Man-Rogosa-Sharp (MRS) supplemented with 0.05% (w/v) L-cysteine hydrochloride and incubated at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, cell pellets/tissue materials were resuspended in 1 ml of QUIAZOL (Qiagen, UK) and placed in a tube containing 0.8 g of glass beads (diameter, 106 μm; Sigma). The cells were lysed by shaking the mix on a BioSpec homogenizer at 4°C for 2 min (maximum setting). The mixture was then centrifuged at 12,000 rpm for 15 min, and the upper phase containing the RNA-containing sample was recovered. The RNA sample was further purified by phenol extraction and ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription and amplification of 500 ng of total RNA was performed with ImProm-IITM Reverse Transcriptase (Promega, Madison, USA) according to the manufacturer’s instructions. 5 µg of RNA was then labelled with ULS Labelling kit with Cy5 or Cy3 (Kreatech, The Netherlands). For the purpose of the tiling array, samples containing kefir/kefiran cDNA was labelled with Cy5, while control samples (MRS) were labelled with Cy3.
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA was hybridized using the Agilent Gene Transcription hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Transcription Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent's standard procedures.
|
| Scan protocol |
Scanning was performed on InnoScan 700 (Innopsys) scanner.
|
| Description |
kefiran_II
|
| Data processing |
Signal intensities for each spot and data extraction were carried out using Mapix 5.5 software (Innopsys, Carbonne, France). Data were analyzed for the final version using the median of the values of each ORF. In addition "TMev" software (http://www.tm4.org/mev/documentation/mev.html) has been used for statistical analysis. In particular, an adjustment of the data that has been applied consisting by a median center samples/column, followed by LIMMA software (Linear Models for Microarray data).
Only B. bifidum PRL2010 genes were analyzed (resulting in a subset of 8130 ID_REFs out of a possible 36186).
|
| |
|
| Submission date |
Aug 30, 2013 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Marco Ventura |
| E-mail(s) |
marco.ventura@unipr.it
|
| Phone |
+390521905666
|
| Organization name |
University of Parma
|
| Department |
Dept. Life Sciences
|
| Lab |
Probiogenomics Lab.
|
| Street address |
Viale delle Scienze 11/A
|
| City |
Parma |
| ZIP/Postal code |
43124 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17646 |
| Series (1) |
| GSE50506 |
Molecular interaction of bifidobacteria with kefir grains |
|
