|
| Status |
Public on Dec 22, 2014 |
| Title |
Gene expression in malO mutant biol rpt 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
malO mutant during stationary phase
|
| Organism |
Clostridium perfringens str. 13 |
| Characteristics |
genotype: malO mutant
|
| Treatment protocol |
Gene expression was compared between a malO mutant of C. perfringens strain 13 and wild-type during stationary phase growth
|
| Growth protocol |
Bacterial strains were cultured for 10 hrs at 37˚C in sterile TPYG broth (5 % [w/v] tryptone, 0.5 % [w/v] proteose peptone, 0.3 % [w/v] yeast extract and 0.1 % [w/v] sodium thioglycolate, supplimented with sterile glucose to a final concentration of 0.38 % [v/v] after autoclaving) under anaerobic conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested from 15 ml aliquots of bacterial culture by centrifugation at 5800 x g for 10 mins. Cell pellets were treated with lysozyme (10 mg/ml) for 15 mins prior to RNA extraction using TriZol (Invitrogen) according to the manufacturer's instructions. Repeated rounds to DNase I digestion and Trizol extraction were performed until DNA contaminiation was removed.
|
| Label |
Cy3
|
| Label protocol |
cDNA synthesis and labelling with Cy3 or Cy5 using 4 μg of total RNA was performed according to the Genisphere 3DNA 900MPX kit (Genisphere) according to the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
wild-type strain 13 during stationary phase
|
| Organism |
Clostridium perfringens str. 13 |
| Characteristics |
genotype: wild-type
|
| Treatment protocol |
Gene expression was compared between a malO mutant of C. perfringens strain 13 and wild-type during stationary phase growth
|
| Growth protocol |
Bacterial strains were cultured for 10 hrs at 37˚C in sterile TPYG broth (5 % [w/v] tryptone, 0.5 % [w/v] proteose peptone, 0.3 % [w/v] yeast extract and 0.1 % [w/v] sodium thioglycolate, supplimented with sterile glucose to a final concentration of 0.38 % [v/v] after autoclaving) under anaerobic conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested from 15 ml aliquots of bacterial culture by centrifugation at 5800 x g for 10 mins. Cell pellets were treated with lysozyme (10 mg/ml) for 15 mins prior to RNA extraction using TriZol (Invitrogen) according to the manufacturer's instructions. Repeated rounds to DNase I digestion and Trizol extraction were performed until DNA contaminiation was removed.
|
| Label |
Cy5
|
| Label protocol |
cDNA synthesis and labelling with Cy3 or Cy5 using 4 μg of total RNA was performed according to the Genisphere 3DNA 900MPX kit (Genisphere) according to the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
Microarrays were pre-hybridised in a solution of 25 % formamide, 5X SSC, 0.1 % SDS and 10 mg/ml of herring sperm DNA at 42˚C for 45 mins. Labelled cDNA was hybridised to the prehybridised microarrays according to the instructions provided with the Geneisphere 3DNA 900MPX kit
|
| Scan protocol |
The dried microarrays were scanned using a GMS418 microarray scanner (Affymetrix), scans were aquired using GMS scanner software (V.1.51.0.42) (Affymetrix) and quantified using Imagene version 5.1 (BioDiscovery).
|
| Data processing |
Quantified ImaGene files from four independant microarray experiments were analysed using the LIMMA software package for R. Each microarray experiment was performed using an independant biological replicate, thus providing four independant biological replicates with two dye-swaps. Spot intensities were firstly normalised per print tip group before being normalised between arrays using a Lowess algorithm. Spot intensities were then log2 transformed and expression values calculated as the ratio of log2 intensity of malO mutant/wild-type.
|
| |
|
| Submission date |
Apr 18, 2011 |
| Last update date |
Dec 22, 2014 |
| Contact name |
Julian I Rood |
| E-mail(s) |
[email protected]
|
| Phone |
61 3 9902 9157
|
| Organization name |
Monash University
|
| Department |
Microbiology
|
| Lab |
Rood
|
| Street address |
Wellington Road
|
| City |
Clayton |
| State/province |
Victoria |
| ZIP/Postal code |
3800 |
| Country |
Australia |
| |
|
| Platform ID |
GPL11408 |
| Series (1) |
| GSE28699 |
Transcriptome of a malO deletion mutant of Clostridium perfringens during stationary phase growth. |
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