|
| Status |
Public on Jul 01, 2015 |
| Title |
BR2 |
| Sample type |
RNA |
| |
|
| Source name |
THP-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte-derived macrophage
infected strain: Beijing resistant MTB strain 2
|
| Treatment protocol |
The MTB cells were harvested by centrifugation and the pellet was resuspended in RPMI medium and added to the macrophages. Macrophages were infected at a multiplicity of infection (MOI) of 3 bacilli/cell for 2 hours, and the excess free-floating bacilli were removed by washing the culture with fresh RPMI containing 10µg/ml gentamicin. The culture was incubated in a fresh RPMI medium without antibiotics at 37°C, 5% CO2for 72-hours.
|
| Growth protocol |
THP-1 Cells were incubated with phorbolmyristate acetate (5ng/ml PMA) for 48hours to induce differentiation into a macrophage phenotype. MTB isolates were cultured in Middlebrook7H9 (BD Biosciences) at 370C, 5% CO2 until the cultures reached McFarland 1 (about 107 CFU/mL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from macrophages with the mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX, USA) in accordance with the manufacturer’s instructions.
|
| Label |
FAM
|
| Label protocol |
The TaqMan®MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used for preparation of cDNA.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Test
|
| Data processing |
The cycle threshold (Ct) raw data was analyzed by two manufacturer’s softwares; SDS 2.4 and RQ Manager 1.2.1.
The expression level of each miRNA was calculated by the relative quantity (RQ value) (2-∆∆Ct) method. The internal control, Mammal U6, was selected for normalization across all experiments. The Ct raw data were determined by using an automatic baseline and a threshold of 0.2(RQ Manager, ABI, Life Technologies).
|
| |
|
| Submission date |
Feb 10, 2015 |
| Last update date |
Jul 01, 2015 |
| Contact name |
Margaret Ip |
| E-mail(s) |
margaretip@cuhk.edu.hk
|
| Organization name |
The Chinese University of Hong Kong
|
| Department |
Department of Microbiology
|
| Street address |
Main clinic building, Prince of Whales Hospital, City one, Shatin
|
| City |
Hong Kong |
| ZIP/Postal code |
00852 |
| Country |
China |
| |
|
| Platform ID |
GPL19763 |
| Series (1) |
| GSE65811 |
miRNA profile of THP-1 macrophage infected with different MTB strains |
|
