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Status |
Public on Dec 01, 2012 |
Title |
IST408_bceE::Tp, biological rep3 |
Sample type |
RNA |
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Source name |
IST408 bceE::Tp with 12h growth in S medium
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Organism |
Burkholderia cepacia |
Characteristics |
isolate: IST408 bceE::Tp genotype: bceE mutation
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Treatment protocol |
Bacterial cells were resuspended in RNAprotect Bacteria Reagent (Qiagen).
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Growth protocol |
Bacterial strains were grown in 100 ml S medium in 250 ml Erlenmeyer flasks at 30ºC, 250 r.p.m. for 12 h.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extraction was carried out using the RNeasy MiniKit (Qiagen) by following the manufacturer's recommendations.
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Label |
Biotin
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Label protocol |
RNA was processed for use on Affymetrix custom dual-species Burkholderia arrays according to the manufacturer's Prokaryotic Target Preparation Assay. Briefly, 10 ug of total RNA containing spiked-in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix, Santa Clara, CA) was used in a reverse transcription reaction with random primers (Invitrogen Life Technologies) to generate first-strand cDNA. After removal of RNA, 2 ug of cDNA were fragmented with DNase and end-labeled with biotin using terminal polynucleotidyl transferase (GeneChip WT Terminal Labeling Kit; Affymetrix). Size distribution of the fragmented and end-labeled cDNA was assessed using an Agilent 2100 Bioanalyzer.
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Hybridization protocol |
Following fragmentation, 2 µg of end-labeled fragmented cDNA were used in a 200-µl hybridization cocktail containing added hybridization controls and hybridized on arrays for 16 hours at 50ºC. Modified post-hybridization wash and double-stain protocols (FLEX450_0005; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
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Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
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Description |
IST408_bceE_3 Mutation in the bceE gene.
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Data processing |
DNA-based probe-selection was performed using Xspecies DNA hyb CDF batchmaker v3.2, available here: http://affymetrix.arabidopsis.info/xspecies/. An optimal signal cutoff of 80 and minimum number of probes per probe set of 7 were determined empirically, resulting in the masking of 24223 of the original 226576 probes on the array and the exclusion of 156 probe sets. The new cdf file created (Bcc1sa520656F_Xspecies.CEL80.cdf) was used in DNA-Chip Analyzer 2008 for the subsequent gene expression analyses.
The 9 arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the arrays were used to obtain model-based gene expression indices based on a Perfect Match (PM)-only model. Replicate data (triplicates) for each bacterial isolate was weighted gene-wise by using inverse squared standard error as weights. All genes compared were considered to be differentially expressed if the 90% lower confidence bound of the fold change (LCB) between experiment and baseline was above 1.2 when the bceF::Tp mutant was compared with the wild-type IST408 and 1.6 for the comparison between the bceE::Tp mutant and IST408.
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Submission date |
May 23, 2012 |
Last update date |
Dec 01, 2012 |
Contact name |
Leonilde Morais Moreira |
E-mail(s) |
lmoreira@ist.utl.pt
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Phone |
+351 218419031
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Organization name |
Instituto Superior Tecnico
|
Department |
Bioengineering
|
Lab |
Biological Sciences
|
Street address |
A. Rovisco Pais
|
City |
Lisboa |
ZIP/Postal code |
1049-001 |
Country |
Portugal |
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Platform ID |
GPL15600 |
Series (1) |
GSE38183 |
Expression data from Burkholderia cepacia IST408 bceE and bceF mutants |
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