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| Status |
Public on Dec 01, 2017 |
| Title |
PCR_product_GH11_Ralbus_7 |
| Sample type |
genomic |
| |
|
| Source name |
Bacteria
|
| Organism |
Ruminococcus albus |
| Characteristics |
strain: Ruminococcus albus 7
gut origin: Rumen
|
| Treatment protocol |
No treatment
|
| Growth protocol |
The 7 target genes PCR-amplified were beforhand cloned in plasmid as previously described (Rakotoarivonina et al., 2009; Mirande et al., 2010; Devillard et al., 2003) or in this work using PCRII-TOPO vector (Invitrogen). Escherichia coli (One Shot TOP10 Chemically Competent E. coli, Invitrogen) were transformed with recombinant plasmids. Recombinant cells were cultivated in LB medium with ampicillin (100 μg/mL) or kanamycin (50 µg/mL).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Plasmids were extracted with the QIAprep MiniPrep kit (Qiagen) from 5 mL of a 15 h culture of E. coli. Inserts were sequenced to ensure that cloned DNA fragments had the same sequence than genes used for probe design. Cloned genes were then PCR amplified with the Pfx50 DNA Polymerase (Invitrogen). PCR products were gel purified with the QIAquick PCR purification kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Each PCR products (2 µg) were labelled independently using the ULS aRNA Labelling kit (Kreatech Diagnostics) and Cy3 or Cy5 cyanines following the manufacturer’s recommendations. A degree of labelling between 1.0 and 2.5% was considered as acceptable.
|
| |
|
| Hybridization protocol |
DNA hybridization (50 ng for each PCR product) was performed at 65°C during 24 h following the Agilent protocol ‘Oligonucleotide Array-Based CGH for Genomic DNA Analysis’.
|
| Scan protocol |
Immediately after washing steps, microarray slides were scanned using an Agilent G2505B microarray scanner (Agilent Technologies) with a resolution of 5 μm and the XDR mode (Extended Dynamic Range) set up at 10% and 100% PMT gain.
|
| Description |
PCR product
|
| Data processing |
Fluorescence intensities of oligonucleotide spots were extracted from the scanned images using the Feature Extraction software (V11.0, Agilent Technologies). Then data were processed with dedicated scripts based on C++ and Delphi languages. For each probe, median intensity value of the 3 replicates was conserved and used as the probe signal value. The SNR (Signal-to-Noise Ratio), similar to the detection threshold response (positive hybridization) and corresponding to the probe signal value divided by the local background intensity value, was calculated for each probe. The SNR thresholds were set to 3 for 25-mer probes and 6 for 54-mer probes. Such SNR threshold values ensured a very specific response of the FibroChip, except for genes belonging to very close strains from the same species. Results for 25- and 54-mer probes were treated independently and for each probe type, a gene was considered as detected when 65% of probes were positive. The SNR value of a detected gene was then calculated by the mean of the SNR of all 25- or 54-mer probes targeting this gene, meeting or not the defined SNR threshold.
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| |
|
| Submission date |
Nov 30, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Sophie Comtet-Marre |
| Organization name |
Université Clermont Auvergne, INRA
|
| Lab |
UMR 454 MEDIS
|
| Street address |
Centre Auvergne-Rhône-Alpes
|
| City |
Saint-Genès-Champanelle |
| ZIP/Postal code |
63122 |
| Country |
France |
| |
|
| Platform ID |
GPL24327 |
| Series (1) |
| GSE107550 |
The FibroChip, a functional DNA microarray to monitor cellulolysis and hemicellulolysis activities of rumen microbiota |
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