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Status |
Public on Jul 01, 2015 |
Title |
BS2 |
Sample type |
RNA |
|
|
Source name |
THP-1 cells
|
Organism |
Homo sapiens |
Characteristics |
cell type: Monocyte-derived macrophage infected strain: Beijing sensitive 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.
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|
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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).
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|
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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 |
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