|
| Status |
Public on Mar 31, 2010 |
| Title |
NGS treatment, replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NGS-treated experimental sample
|
| Organism |
Leptospira interrogans |
| Characteristics |
serovar: Copenhageni
strain: L533
treatment: normal guinea pig serum (NGS)
|
| Treatment protocol |
One hundred ml cultures of exponential-phase grown L. interrogans serovar Copenhageni strain L533 were divided equally between 2 tubes and harvested by centrifugation at 8,000 xg for 20 min at room temperature. The cell pellet in each tube was resuspended in 5 ml of either prewarmed EMJH or prewarmed 50% normal guinea pig serum (NGS) in EMJH. After incubation at 37°C for 30 min, 0.5 ml of ice-cold killing buffer (50 mM Tris-HCl, pH 7.5, 15 mg/ml sodium azide, 0.6 mg/ml chloramphenicol) was immediately added to each tube before chilling on ice for 5 min. The NGS- and EMJH-treated cells were harvested by centrifugation at 4°C for 15 min before RNA isolation was performed.
|
| Growth protocol |
L. interrogans serovar Copenhageni strain L533 was grown in EMJH broth medium at 30°C under aerobic conditions. Leptospires were grown to exponential phase at an approximate density of 5-8x108cells/ml before harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol (Invitrogen) following manufacturer's instructions and then treated with DNase (20 U for 30 min at 37°C). RNA was further purified using QIAGEN RNeasy minicolumns with on-column DNase treatment according to the manufacturer’s protocol.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Labeled cDNA probes were synthesized from 2.5 micrograms of total RNA using random primers contained in the 3DNA Array 900MPX expression array detection kit (Genisphere) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
EMJH-treated control sample
|
| Organism |
Leptospira interrogans |
| Characteristics |
serovar: Copenhageni
strain: L533
treatment: EMJH broth medium
|
| Treatment protocol |
One hundred ml cultures of exponential-phase grown L. interrogans serovar Copenhageni strain L533 were divided equally between 2 tubes and harvested by centrifugation at 8,000 xg for 20 min at room temperature. The cell pellet in each tube was resuspended in 5 ml of either prewarmed EMJH or prewarmed 50% normal guinea pig serum (NGS) in EMJH. After incubation at 37°C for 30 min, 0.5 ml of ice-cold killing buffer (50 mM Tris-HCl, pH 7.5, 15 mg/ml sodium azide, 0.6 mg/ml chloramphenicol) was immediately added to each tube before chilling on ice for 5 min. The NGS- and EMJH-treated cells were harvested by centrifugation at 4°C for 15 min before RNA isolation was performed.
|
| Growth protocol |
L. interrogans serovar Copenhageni strain L533 was grown in EMJH broth medium at 30°C under aerobic conditions. Leptospires were grown to exponential phase at an approximate density of 5-8x108cells/ml before harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol (Invitrogen) following manufacturer's instructions and then treated with DNase (20 U for 30 min at 37°C). RNA was further purified using QIAGEN RNeasy minicolumns with on-column DNase treatment according to the manufacturer’s protocol.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Labeled cDNA probes were synthesized from 2.5 micrograms of total RNA using random primers contained in the 3DNA Array 900MPX expression array detection kit (Genisphere) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
The microarray was prehybridized for 45 min at 50°C using a solution containing 25% formamide, 5x SSC, 0.1% sodium dodecyl sulfate, 10 mg/ml bovine serum albumin, and 1 mg/ml sheared herring sperm DNA. Hybridization with the cDNA hybridization mix at 50°C overnight and 3DNA hybridization mix at 50°C for 4 h was performed following 3DNA Array 900MPX detection kit (Genisphere) protocol. Slides were washed in ultrapure water and dried by centrifugation at 1,000 xg for 3 min prior to scanning.
|
| Scan protocol |
Scanned on GMS model 418 microarray scanner (Genetic Microsystems). The spot intensities were quantitated with ImaGene version 5.1 (Biodiscovery).
|
| Description |
Analysis used RNA of EMJH-treated control samples for comparison to those of NGS-treated experimental samples.
|
| Data processing |
The web-based program Bioarray Software Environment (BASE) was used for data analysis. Spot-specific median background intensities were subtracted from spot-specific median signals. Only spots with a corrected intensity of greater than 250 were further analyzed. Data normalization for each array was performed independently using the global median ratio, which scales the intensities such that the median of the ratio between Cy3 and Cy5 channels was 1 and spots within 5% of the lowest and the highest intensities were excluded. Print-tip loess normalization was applied to each array, followed by between-arrays normalization, which scales all replicate arrays such that they had the same median absolute deviation. Direct comparison of gene expression between NGS- and EMJH-treated samples was based on moderated t test and associated P values adjusted for multiple testing by controlling the false discovery rate. Differentially expressed genes were considered to be statistically significant if an absolute relative ratio (NGS/EMJH) was greater than 1.5 fold with an adjusted P value of less than 0.01.
For data analysis, median data before normalization with global median ratio, LIMMA+SMA (bioconductor, print-tip Loess), and LIMMA+SMA (between arrays) were selected.
|
| |
|
| Submission date |
Sep 02, 2009 |
| Last update date |
Jan 11, 2010 |
| Contact name |
Kanitha Patarakul |
| E-mail(s) |
[email protected]
|
| Phone |
662-256-4132
|
| Fax |
662-252-5952
|
| Organization name |
Faculty of Medicine, Chulalongkorn University
|
| Department |
Microbiology
|
| Street address |
1873 Rama IV Road
|
| City |
Pathumwan |
| State/province |
Bangkok |
| ZIP/Postal code |
10330 |
| Country |
Thailand |
| |
|
| Platform ID |
GPL9140 |
| Series (1) |
| GSE17942 |
Global transcriptomic response of Leptospira interrogans serovar Copenhageni upon exposure to serum |
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