|
| Status |
Public on Feb 01, 2019 |
| Title |
M68_vs_WT_3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S1H, wildtype
|
| Organism |
Rhodospirillum rubrum |
| Characteristics |
strain: S1H
|
| Treatment protocol |
All strains were grown in normal conditions, as the aim of this study was to compare the general gene expression of two different strains.
|
| Growth protocol |
R. rubrum cultures of S1H and M68 were grown to mid-log phase (ca. OD680 2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Twenty ml of cultures were mixed with RNAprotect® Bacteria Reagent (Qiagen) for total RNA stabilization following manufacturer’s instructions and stored as pellets at -80 °C until further use. Bacterial lysis was done following the protocol for Gram-negative bacteria of the SV Total RNA Isolation System kit (Promega). Briefly, stabilized bacterial pellets were resuspended in 100 μl of freshly prepared TE (Tris 10 mM, EDTA 1 mM) containing lysozyme 3 mg/ml and incubated for 10 minutes at room temperature. Total RNA isolation was performed using SV total RNA Isolation system (Promega). RNA quality/integrity was assessed using a BioAnalyzer 2100 (Agilent) where samples with a RNA Integrity Number (RIN) value above 8 were used for microarray experiment. RNA quantity was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop technologies).
|
| Label |
Cy3
|
| Label protocol |
Ten micrograms of RNA were reverse transcribed using Pronto kit (Promega, United States) following manufacturer’s instructions. Two color labeling, green for solvent control samples and red for condition (Triclosan challenge) samples, was performed using respectively Cy3-dCTP and Cy5-dCTP nucleotides (Amersham BioSciences, United Kingdom).
|
| |
|
| Channel 2 |
| Source name |
M68, QS-deficient mutant
|
| Organism |
Rhodospirillum rubrum |
| Characteristics |
strain: M68
|
| Treatment protocol |
All strains were grown in normal conditions, as the aim of this study was to compare the general gene expression of two different strains.
|
| Growth protocol |
R. rubrum cultures of S1H and M68 were grown to mid-log phase (ca. OD680 2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Twenty ml of cultures were mixed with RNAprotect® Bacteria Reagent (Qiagen) for total RNA stabilization following manufacturer’s instructions and stored as pellets at -80 °C until further use. Bacterial lysis was done following the protocol for Gram-negative bacteria of the SV Total RNA Isolation System kit (Promega). Briefly, stabilized bacterial pellets were resuspended in 100 μl of freshly prepared TE (Tris 10 mM, EDTA 1 mM) containing lysozyme 3 mg/ml and incubated for 10 minutes at room temperature. Total RNA isolation was performed using SV total RNA Isolation system (Promega). RNA quality/integrity was assessed using a BioAnalyzer 2100 (Agilent) where samples with a RNA Integrity Number (RIN) value above 8 were used for microarray experiment. RNA quantity was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop technologies).
|
| Label |
Cy5
|
| Label protocol |
Ten micrograms of RNA were reverse transcribed using Pronto kit (Promega, United States) following manufacturer’s instructions. Two color labeling, green for solvent control samples and red for condition (Triclosan challenge) samples, was performed using respectively Cy3-dCTP and Cy5-dCTP nucleotides (Amersham BioSciences, United Kingdom).
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA was re-suspended in the universal hybridization buffer (Pronto kit), mixed and added to the spotted slide for overnight hybridization at 42°C in a Tecan HS4800 Pro hybridization station (Tecan Group Ltd, Switzerland). Afterwards, the slide was washed according to Pronto kit's protocol.
|
| Scan protocol |
Slides were scanned (at 532 and 635 nm) using the GenePix Personal 4100A microarray scanner (Molecular Devices, USA).
|
| Description |
Comparison of S1H and M68 strain
|
| Data processing |
Micro-array spot-signals were analyzed using the GenePix Pro v.6.0.1 software and flagged according to build-in quality criteria. Raw median intensity data were imported into R version 2.7.0 for statistical analysis using the LIMMA package version 2.15.15 (Smyth 2005) as available from BioConductor. Raw data were background-corrected based on convolution of normal and exponential distributions with an offset of 50 (Ritchie et al. 2007). Data were normalized within each array using the printing-tip loess normalization algorithm (Smyth and Speed 2003). The in-slide replicate correlations were calculated using the Duplicate correlation function in the LIMMA package (Smyth 2005). The log expression values were fitted to al linear model and moderated t-statistics were calculated using empirical Bayes method (Smyth 2004). P-values were corrected for multiple testing using the Benjamin and Hochberg’s method to control the false discovery rate (Benjamini and Hochberg 1995).
|
| |
|
| Submission date |
Jan 08, 2016 |
| Last update date |
Feb 01, 2019 |
| Contact name |
Rob Van Houdt |
| E-mail(s) |
[email protected]
|
| Phone |
+3214332728
|
| Organization name |
SCK-CEN
|
| Department |
Interdisciplinary Biosciences
|
| Lab |
Microbiology Unit
|
| Street address |
Boeretang 200
|
| City |
Mol |
| ZIP/Postal code |
2400 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL7256 |
| Series (1) |
| GSE76666 |
Transcriptomic and proteomic profiling of Rhodospirillum rubrum M68 cultivated in MELiSSA related culture conditions |
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