|
| Status |
Public on Nov 11, 2010 |
| Title |
MG1363 versus NZ9000 in CDM |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MG1363
|
| Organism |
Lactococcus cremoris subsp. cremoris MG1363 |
| Characteristics |
culture medium: CDM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from two replicate cultures of both L. lactis ΔlmrCD and cholate-adapted L. lactis ΔlmrCDR. Cultures were grown at 30 ºC in GM17 medium and cells were harvested at the OD660 of ~ 1.RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (PMID: 17085554). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (PMID: 17085554 and 15907200). In all cases, dye-swapping was performed.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions.
|
| |
|
| Channel 2 |
| Source name |
NZ9000
|
| Organism |
Lactococcus cremoris subsp. cremoris NZ9000 |
| Characteristics |
culture medium: CDM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from two replicate cultures of both L. lactis ΔlmrCD and cholate-adapted L. lactis ΔlmrCDR. Cultures were grown at 30 ºC in GM17 medium and cells were harvested at the OD660 of ~ 1.RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (PMID: 17085554). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (PMID: 17085554 and 15907200). In all cases, dye-swapping was performed.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions.
|
| |
|
| |
| Hybridization protocol |
Hybridization (16 h at 45 °C) of labeled cDNA was performed in Ambion Slidehyb #1 hybridization buffer (Ambion Europe) on superamine glass slides (Array-It; SMMBC), containing technical replicates.
|
| Scan protocol |
Slides were scanned using the Genepix 4200AL DNA micro array slide scanner.
Quantification of the signal of the spots on slides was done with ArrayPro 4.5.
|
| Description |
Hybridization of strain MG1363 and NZ9000 grown in CDM
|
| Data processing |
Slide data were processed by using MicroPreP as described previously (10). Prior to the analysis, automatically and manually flagged spots and spots with very low background-subtracted signal intensities (5% of the weakest spots [sum of Cy3 and Cy5 net signals]) were filtered out. Net signal intensities were calculated by using grid-based background subtraction. In postprep, negative and empty values were eliminated, and outliers were removed by the deviation test. Differential expression tests were performed by using a Cyber-T Student's t test for paired data (19).
|
| |
|
| Submission date |
May 10, 2010 |
| Last update date |
Nov 11, 2010 |
| Contact name |
Anne de Jong |
| E-mail(s) |
[email protected]
|
| Phone |
+31 50 363 2047
|
| Organization name |
university of Groningen
|
| Department |
Molecular Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL5048 |
| Series (1) |
| GSE21759 |
Genome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies |
|
