|
| Status |
Public on Jul 01, 2015 |
| Title |
Latent2 |
| Sample type |
RNA |
| |
|
| Source name |
whole blood
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte-derived macrophage
age: 57
gender: Female
infection status: Latent TB patient
|
| Growth protocol |
PBMCs were freshly harvested from the patients’ whole blood by using the Ficoll-Hypaque column (GE healthcare).The PBMC was resuspended in ice-cold monocyte adhesion medium (RPMI1640 + 7.5% autologous plasma, 1% penicillin-streptomycin) and incubated in a petri dish for 90minutes at 370C. The adherent monocytes were washed with warm RPMI medium several times to remove loosely attached cells. The monocytes were detached by incubation with PBS containing 5 mM EDTA for 10–20 minutes at room temperature and were collected by centrifugation. The monocytes were refed by fresh medium every 2 days and allowed to differentiate into macrophages for 10 days in vitro.
|
| 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) |
| GSE65810 |
miRNA profile of macrophage from different TB patients |
|
