|
| Status |
Public on Aug 04, 2020 |
| Title |
IPF LF-derived EVs |
| Sample type |
RNA |
| |
|
| Source name |
EVs collected from LFs from patients with IPF
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: idiopathic pulmonary fibrosis (IPF) patients
cell type: Primary lung fibroblast (LFs)
sample type: Extracellular vesicles (EVs)
molecule subtype: miRNA
|
| Treatment protocol |
The IPF and non-IPF LFs were washed with phosphate-buffered saline (PBS), and the culture medium was replaced with serum-free advanced DMEM medium contained antibiotic and antifungal drugs. After incubation, the conditioned medium (CM) was collected and centrifuged at 2,000 x g for 10 min at 4°C. To thoroughly remove cellular debris, the supernatant was filtered through a 0.22-mm filter (Millipore). The CM was then used for EV isolation. To prepare EVs, CM was ultracentrifuged at 35,000 rpm using a SW41Ti rotor for 70 min at 4°C. The pellets were washed with PBS, ultracentrifuged at 35,000 rpm using the SW41Ti rotor for 70 min at 4°C and resuspended in PBS.
|
| Growth protocol |
Primary lung fibroblasts (LFs) were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, CA, USA) with 10% fetal bovine serum and 100 μg/ml penicillin/streptomycin and 0.5 mM sodium pyruvate (Sigma-Aldrich, MO, USA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using QIAzol and the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocols. RNA was quantified using a NanoDrop-1000 spectrophotometer and a Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.).
|
| Label |
Cy3
|
| Label protocol |
The miRNA Complete Labeling and Hyb Kit (Agilent Technologies)
|
| |
|
| Hybridization protocol |
Agilent one-color gene expression hyb/wash protocol
|
| Scan protocol |
Agilent SureScan G4900DA
|
| Description |
Total RNA extracted form extracellular vesicles, and 20 ng of RNA for analysis
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jan 04, 2019 |
| Last update date |
Aug 04, 2020 |
| Contact name |
Tsukasa Kadota |
| E-mail(s) |
tkskdt@jikei.ac.jp
|
| Organization name |
The Jikei University School of Medicine
|
| Department |
Division of Respiratory Diseases, Department of Internal Medicine
|
| Street address |
3-19-18, Nishishimbashi, Minato-ku
|
| City |
Tokyo |
| ZIP/Postal code |
105-0003 |
| Country |
Japan |
| |
|
| Platform ID |
GPL25134 |
| Series (1) |
| GSE124665 |
The profile of miRNAs in lung fibroblast-derived extracellular vesicles from idiopathic pulmonary fibrosis (IPF) patients or non-IPF individuals. |
|
