|
| Status |
Public on Oct 01, 2010 |
| Title |
THP-1 Cells: Uninfected vs. C. burnetii NMII infected - bio rep1, tech rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA was harvested from Uninfected THP-1 human monocytic leukemia cells
|
| Organism |
Homo sapiens |
| Characteristics |
infection: uninfected
cell line: THP-1(TIB-202)
passage: < 8
cell type: Monocytic leukemia cells derived from peripheral blood
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Non-adherent THP-1 human monocytic leukemia cells (TIB-202; ATCC) were propagated in RPMI 1640 medium (Gibco, Carlsbad, CA) supplemented with 1mM sodium pyruvate, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions. Total RNA (500 ng) from each sample was then amplified using an Epicentre® Biotechnologies (Madison, WI) TargetAmp™ 1-Round AminoallylaRNA Amplification Kit, yielding approximately 6-10μg of aminoallyl-aRNA (AA-aRNA).
|
| Label |
Alexa Fluor® 555-GREEN
|
| Label protocol |
Approximately 6-10μg of aminoallyl-aRNA (AA-aRNA) was used for labeling. Alexa Fluor® 555-GREEN (Invitrogen, Carslbad, CA) was used to label the uninfected AA-aRNA and CAM treated uninfected AA-aRNA, while Alexa Fluor® 647-RED (Invitrogen) was used to label the AAaRNA from the C. burnetii infected cells and also for the CAM treated AAaRNA from the C. burnetii infected cells.
|
| |
|
| Channel 2 |
| Source name |
Total RNA was harvested from C.burnetii infected THP-1 human monocytic leukemia cells
|
| Organism |
Homo sapiens |
| Characteristics |
infection: C. burnetii infected
cell line: THP-1(TIB-202)
passage: < 8
cell type: Monocytic leukemia cells derived from peripheral blood
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Non-adherent THP-1 human monocytic leukemia cells (TIB-202; ATCC) were propagated in RPMI 1640 medium (Gibco, Carlsbad, CA) supplemented with 1mM sodium pyruvate, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2.Purified C. burnetii NMII SCVs at a genome equivalent MOI of 15 were used to establish a synchronous infection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions. Total RNA (500 ng) from each sample was then amplified using an Epicentre® Biotechnologies (Madison, WI) TargetAmp™ 1-Round AminoallylaRNA Amplification Kit, yielding approximately 6-10μg of aminoallyl-aRNA (AA-aRNA).
|
| Label |
Alexa Fluor® 647-RED
|
| Label protocol |
Approximately 6-10μg of aminoallyl-aRNA (AA-aRNA) was used for labeling. Alexa Fluor® 555-GREEN (Invitrogen, Carslbad, CA) was used to label the uninfected AA-aRNA and CAM treated uninfected AA-aRNA, while Alexa Fluor® 647-RED (Invitrogen) was used to label the AAaRNA from the C. burnetii infected cells and also for the CAM treated AAaRNA from the C. burnetii infected cells.
|
| |
|
| |
| Hybridization protocol |
Labeled AA-aRNA (2μg) with a dye incorporation efficiency range of 18 -34 picomol/microgram, were mixed pair-wise and hybridized overnight to Human OneArray™ microarrays (Phalanx Biotech Group, Palo Alto, CA). Human OneArrays contain 32,050 oligonucleotides; 30968 human genome probes and 1082 experimental control probes formed as 60-mer sense-strand DNA elements. Arrays were hybridized, washed and dried rapidly according to the manufacturer’s instructions.
|
| Scan protocol |
Signal intensity of the hybridized arrays were measured by ScanArray Express (PerkinElmer, Boston, MA, USA) and the images were processed using GenePix Pro version 4.0 (Axon, Union City, CA, USA)
|
| Description |
Biological replicate 1 of 3 - Technical replicate 2.
|
| Data processing |
The processed GenePix Pro 4.0 output was further analyzed using Loess-Global intensity dependent normalization through the GenePix Auto Processor (http://darwin.biochem.okstate.edu/gpap3/)
|
| |
|
| Submission date |
Aug 17, 2010 |
| Last update date |
Aug 24, 2010 |
| Contact name |
SAUGATA MAHAPATRA |
| E-mail(s) |
saugata.mahapatra@okstate.edu
|
| Phone |
405-744-2506
|
| Fax |
405 744 6790
|
| URL |
http://microgenetics.okstate.edu/~eshaw/
|
| Organization name |
OKLAHOMA STATE UNIVERSITY
|
| Department |
MICROBIOLOGY AND MOLECULAR GENETICS
|
| Lab |
OBLIGATE INTRCELLULAR PATHOGENESIS
|
| Street address |
307 LSE, OSU
|
| City |
STILLWATER |
| State/province |
OK |
| ZIP/Postal code |
74078 |
| Country |
USA |
| |
|
| Platform ID |
GPL6254 |
| Series (1) |
| GSE23665 |
Human THP-1 Cells: Uninfected vs. Coxiella burnetii NMII infected compared to Uninfected with Chloramphenicol vs. Coxiella burnetii NMII infected with Chloramphenicol |
|
