|
| Status |
Public on Dec 31, 2011 |
| Title |
In_vitro_bac_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
In vitro bacteria
|
| Organism |
Burkholderia pseudomallei |
| Characteristics |
clinical isolate: D286
|
| Treatment protocol |
Infected macrophage cells were lysed with 1% saponin in PBS at 37°C for 5 min and cells lysate were subjected to centrifugation. Bacterial pellet collected were snap-frozen in liquid nitrogen and kept at -80°C till extraction. Similar treatments were also done for control bacteria.
|
| Growth protocol |
Prior to infection, overnight bacteria cultures were diluted to 1:50 in LB broth and grown to mid-logarithmic phase (OD600 = 0.4-0.6) at 37°C, 250 rpm for 3 h. Human macrophage U937 cells were then infected at MOI of 10:1 and intracellular bacteria were harvested at selected time post-infection. For control, bacteria were grown at stationary in complete RPMI medium under 5% CO2 till mid-logarithmic phase before RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial RNA was prepared using the Qiagen’s RNeasy Mini Kit and on-column DNase I digestion was performed. Total RNA obtained was purified further by ethanol/ammonium acetate precipitation. Quality control was performed with Agilent 2100 Bioanalyser.
|
| Label |
Cy3
|
| Label protocol |
Prior to labelling, bacteria total RNA were polyadenylated based on the PAP method using A-plusTM Poly(A) Polymerase Tailing kit (Epicentre). 500 ng of polyadenylated RNA was reverse transcribed to cRNA and labelled with Agilent’s Low RNA Input Linear Amplification kit according to the manufacturer's instruction.
|
| |
|
| Hybridization protocol |
Standard Agilent One-colour Microarray hybridization protocol
|
| Scan protocol |
Arrays were scanned with the Agilent Technologies Scanner model G2505B according to Agilent One-colour microarray scanning protocol.
|
| Description |
Biological replicate 2
|
| Data processing |
Spot intensities and other quality control features were extracted with Agilent’s Feature Extraction Software version 9.5.3.1. Processed signals from Feature Extraction were used as spot intensities for analysis and gIsWellAboveBG is used to flag non-significant features with background-subtracted signal is lower than 2.6 times the background standard deviation of that feature. Arrays were then median normalized with BRB-ArrayTools (http://linus.nci.nih.gov).
|
| |
|
| Submission date |
Feb 28, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Sheila Nathan |
| E-mail(s) |
snathan2005@yahoo.co.uk
|
| Phone |
+60389267446
|
| Fax |
+60389252698
|
| Organization name |
Universiti Kebangsaan Malaysia
|
| Department |
Faculty of Science & Technology
|
| Lab |
Block 5B
|
| Street address |
Jalan Universiti
|
| City |
Bangi |
| State/province |
Selangor |
| ZIP/Postal code |
43600 |
| Country |
Malaysia |
| |
|
| Platform ID |
GPL13233 |
| Series (1) |
| GSE27558 |
Burkholderia pseudomallei transcriptional adaptation in macrophage |
|
