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Status |
Public on Jul 29, 2015 |
Title |
Escherichia coli O157:H7 EDL933, 0.005 mg/ml of Ginkgolic acid |
Sample type |
RNA |
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Source name |
Escherichia coli O157:H7 EDL933
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Organism |
Escherichia coli O157:H7 str. EDL933 |
Characteristics |
treatment: 0.005 mg/ml Ginkgolic acid
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Treatment protocol |
Sample 2 was incubated with 0.005 mg/ml of Ginkgolic acid at 37oC for 8 hrs with 250 rpm shaking
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Growth protocol |
E. coli was inoculated in 25 ml of LB medium in 250 ml shaker flasks with overnight cultures (1 : 100 dilution). Cells were cultured for 8 h with shaking at 250 rpm with and without ginkgolic acid (0.005 mg/ml)
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Extracted molecule |
total RNA |
Extraction protocol |
DNA microarray analysis with one biological replicate was performed with an Affymetrix system. E. coli was inoculated in 25 ml of LB medium in 250 ml shaker flasks with overnight cultures (1 : 100 dilution). Cells were cultured for 8 h with shaking at 250 rpm with and without 0.005 mg/ml of ginkgolic acid. Before sample collection, RNase inhibitor (RNAlater, Ambion, TX, USA) was added, and the cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 s before centrifugation at 16,000 g for 2 min. The cell pellets were immediately frozen with dry ice and stored at –80°C. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA) with Qiagen RNase-free DNase I (Cat# 79254).
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Label |
biotin
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Label protocol |
Following affymetrix protocol. cDNA was synthesized first using Promega M-MLV Reverse transcriptase (cat# M1705). After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181).
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Hybridization protocol |
Following affymetrix protocol. Prepared hybridization cocktail for Single Probe Array (49 Format) with total 200 ul volume including 1X hybrization buffer, 50 pM B2 Control Oligo, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, and at least 1 ug fragmented and labelled cDNA. After loading of hybridization cocktail in Affymetrix E. coli Antisense Genome Array, the hybridization was performed at 45ºC, with 60 rpm for 16 hours. After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS).
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Scan protocol |
Following affymetrix protocol. After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS).
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Description |
Gene expression data of E. coli O157:H7 incubated with 0.005 mg/ml of Ginkgolic acid for 8 hrs at 37oC with 250 rpm shaking
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Data processing |
The data were analyzed with Robust Multi-array Average (RMA) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized, and log transformed intensity values were then analyzed using GeneSpring GX 12.5 (Agilent technologies, CA). Fold change filters included the requirement that the genes be present in at least 150% of controls for up-regulated genes and lower than 66% of controls for down-regulated genes. Hierarchical clustering data were clustered groups that behave similarly across experiments using GeneSpring GX 12.5 (Agilent technologies, CA). Clustering algorithm was Euclidean distance, average linkage.
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Submission date |
Jul 30, 2013 |
Last update date |
Jul 29, 2015 |
Contact name |
Jintae Lee |
E-mail(s) |
jtlee@ynu.ac.kr
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Phone |
82-53-810-2533
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Organization name |
Yeungnam University
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Department |
Chemical engineering
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Lab |
Biotechnology
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Street address |
214-1 Daedong
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City |
Gyeongsan-Si |
State/province |
Gyeongsangbuk-Do |
ZIP/Postal code |
712-749 |
Country |
South Korea |
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Platform ID |
GPL3154 |
Series (1) |
GSE49367 |
Ginkgolic acids and Ginko biloba extract inhibit Escherichia coli O157:H7 biofilm formation |
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