|
| Status |
Public on Sep 28, 2012 |
| Title |
Normal fibroblast NF2 |
| Sample type |
RNA |
| |
|
| Source name |
Normal fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Normal fibroblast
gender: female
disease state: breast cancer
source of cells: specimen 2
|
| Treatment protocol |
Tissues were washed 3 times with PBS containing 100U/ml penicillin and 100 ug/ml streptomycin(Sigma, St. Louis, MO, USA), then minced into small pieces and digested for 8 h at 37°C in DMEM containing 10% FBS and 0.5 mg/ml collagenase І (Sigma, St. Louis, MO, USA). After centrifugation and washing with PBS, cells pellets were resuspended in the DMEM with 10% FBS and transfered into 100-mm tissue-culture dishes. CAFs and NFs were routinely maintained in DMEM containing 10% FBS at 37°C in humidified atmosphere containing 5% CO2. The third passage of primary cells was used in the experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the mir-VanaTM miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. The RNA quantity was determined using Nanodrop1000 (Thermo Fisher Scientific, Waltham, MA, USA) and the RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All RNA samples revealed acceptable RNA Integrity Number (RIN) values between 8.8 and 9.6.
|
| Label |
CY3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using Agilent’s miRNA Complete Labeling and Hyb Kit (Agilent) according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA was hybridized to Homo sapiens Agilent-021827 Human miRNA Microarray G4470C (Feature Number version) using Agilent’s miRNA Complete Labeling and Hyb Kit (Agilent) according to the manufacturer's instructions.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k formates (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to Hi 100% and Lo 5%).
|
| Description |
The third passage of primary normal fibroblast cells was used in the experiments.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters (protocol miRNA-v1_95_May07 and Grid: Online Update) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 23, 2012 |
| Last update date |
Sep 28, 2012 |
| Contact name |
liuyang zhao |
| E-mail(s) |
wszly626@gmail.com
|
| Phone |
86-23-68485184
|
| Organization name |
Chongqing Medical University
|
| Street address |
1# Yixueyuan Road, Yuzhong District
|
| City |
Chongqing |
| ZIP/Postal code |
400016 |
| Country |
China |
| |
|
| Platform ID |
GPL14767 |
| Series (1) |
| GSE37527 |
MiRNA expression analysis of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) in breast cancer |
|
