|
| Status |
Public on Jun 10, 2016 |
| Title |
RA FLSs 3 |
| Sample type |
RNA |
| |
|
| Source name |
fibroblast-like synoviocytes from rheumatoid arthritis patient
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: rheumatoid arthritis patient
Sex: female
age: 49 years
cell type: fibroblast-like synoviocytes
|
| Growth protocol |
Tissue specimens were minced into small pieces and digested for 2 hours with 2 mg/ml of type II collagenase (Invitrogen, Carlsbad, CA, USA) in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) at 37°C. After centrifugation at 210 × g for 5 minutes, the precipitate was resuspended with 1 ml of high-glucose DMEM containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 units/ml streptomycin, and then cultured in 25-cm2 cell culture flasks (Corning) in a humidified 5% CO2 incubator. After 10 hours, 4 ml of high-glucose DMEM containing 10% FBS was added to the cell culture flask. All experiments were conducted using cells at passage 3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from fibroblast-like synoviocytes using Agient total RNA isolation Mini kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacture's recommended protocol. The quality and the concentration of the RNA samples were monitored at absorbance ratios of A260/A280 and A260/A230 using a NanoDrop 8000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × blocking agent and 1 μl of 25 × fragmentation buffer, heated to 60 °C for 30 min, and diluted with 25 μl 2 × GE hybridization buffer. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the Human LncRNA Array v3.0 slide (8 x 60K, Arraystar). The slides were incubated for 17 hours at 65°C in an Agilent hybridization oven then washed, fixed and scanned.
|
| Scan protocol |
The slides were scanned using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
lncRNAs and mRNAs expression
RA 3
|
| Data processing |
The scanned were images were analysed using the Agilent Feature Extraction software version 11.0.1.1. Data normalization was carried out using Agilent GeneSpring GX. After quantile normalization of the raw data, LncRNAs and mRNAs that at least 1 out of 6 samples have flags in Present or Marginal were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs between two groups were identified through Volcano Plot filtering.
|
| |
|
| Submission date |
Jun 09, 2016 |
| Last update date |
Jun 10, 2016 |
| Contact name |
Yu Zhang |
| E-mail(s) |
zhangyu00110011@126.com
|
| Phone |
+86-15751011866
|
| Organization name |
Jinling Hospital
|
| Department |
Clinical Laboratory Science
|
| Street address |
305 East Zhongshan Road
|
| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210002 |
| Country |
China |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE83147 |
RNA expression profiles in fibroblast-like synoviocytes from rheumatoid arthritis patients and trauma patients |
|
