|
| Status |
Public on Dec 31, 2016 |
| Title |
AE126_vs_AE126_ZnResist3_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AE126
|
| Organism |
Cupriavidus metallidurans |
| Characteristics |
strain: AE126
|
| 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 |
The strains were cultivated by inoculating 30 ml of MM284 in biological triplicates with 300 μL of a stationary phase culture at 30 °C. These subcultures were allowed to grow until an OD600 value of around 0.6 was reached. Next, cells were harvested by centrifugation for 2 min at 10000 rpm and the bacterial pellets were flash frozen by immersion into liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The bacterial pellets were kept frozen at -80 °C until total RNA extraction was performed using the SV Total RNA Isolation system (Promega Corporation). The quantity of extracted RNA was measured using a NanoDropTM 1000 spectrophotometer (Thermo Scientific). The RNA quality was determined with a Bioanalyzer (Agilent 2100 Electrophoresis Bioanalyzer Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Ten micrograms of RNA were reverse transcribed following the instructions provided with the Pronto kit (Promega, United States). Two color labeling, green for control samples and red for condition (metal challenge) samples, was performed using respectively CyTM3-dCTP and CyTM5-dCTP nucleotides (Amersham BioSciences, United Kingdom).
|
| |
|
| Channel 2 |
| Source name |
AE126_ZnResist_3
|
| Organism |
Cupriavidus metallidurans |
| Characteristics |
strain: AE126_ZnResist_3
|
| 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 |
The strains were cultivated by inoculating 30 ml of MM284 in biological triplicates with 300 μL of a stationary phase culture at 30 °C. These subcultures were allowed to grow until an OD600 value of around 0.6 was reached. Next, cells were harvested by centrifugation for 2 min at 10000 rpm and the bacterial pellets were flash frozen by immersion into liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The bacterial pellets were kept frozen at -80 °C until total RNA extraction was performed using the SV Total RNA Isolation system (Promega Corporation). The quantity of extracted RNA was measured using a NanoDropTM 1000 spectrophotometer (Thermo Scientific). The RNA quality was determined with a Bioanalyzer (Agilent 2100 Electrophoresis Bioanalyzer Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Ten micrograms of RNA were reverse transcribed following the instructions provided with the Pronto kit (Promega, United States). Two color labeling, green for control samples and red for condition (metal challenge) samples, was performed using respectively CyTM3-dCTP and CyTM5-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 AE126 with AE126 zinc resistant strain 3 from lab evolution experiment
|
| 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 |
Oct 16, 2015 |
| Last update date |
Dec 31, 2016 |
| Contact name |
Rob Van Houdt |
| E-mail(s) |
rvhoudto@sckcen.be
|
| 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 |
GPL4980 |
| Series (1) |
| GSE74091 |
Zinc-Induced Transposition of Insertion Sequence Elements Contributes to Increased Adaptability of Cupriavidus metallidurans |
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