|
| Status |
Public on Jan 28, 2016 |
| Title |
lncRNA_Quiescent human aortic vascular smooth muscle cells (HAVSMCs) 1 |
| Sample type |
RNA |
| |
|
| Source name |
Human aortic vascular smooth muscle cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human aortic vascular smooth muscle cells
growth state: quiescent
|
| Biomaterial provider |
ScienCell
|
| Treatment protocol |
Recombinant human platelet-derived growth factor-BB(PDGF-BB) was obtained from R&D Systems Inc. HAVSMCs were treated with 10ng/ml PDGF-BB for 24h.
|
| Growth protocol |
Smooth Muscle Cell Medium (ScienCell, consists of 500 ml of basal medium, 2 ml of fetal bovine serum (Cat. No. 0010), and 5 ml of penicillin/streptomycin solution (Cat. No. 0503) , 37℃, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from three proliferative HAVSMCs(10ng/ml PDGF-BB-treated for 24h) and quiescent HAVSMCs (untreated for 24h) using Trizol reagent (Invitrogen), according to the manufacturer's protocol.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). 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, then heated the mixture at 60°C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed, and scanned by the Agilent Scanner G2505C.
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 3 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through P-value/FDR filtering. Differentially expressed LncRNAs and mRNAs between the two samples were identified through Fold Change filtering. Hierarchical Clustering and combined analysis were performed using homemade scripts.
|
| |
|
| Submission date |
Jan 27, 2016 |
| Last update date |
Mar 15, 2016 |
| Contact name |
shaoguang sun |
| E-mail(s) |
sunshaog@hebmu.edu.cn
|
| Phone |
8631186265639
|
| Organization name |
Hebei medical university
|
| Department |
biochemistry and molecular biology
|
| Street address |
zhong shan dong lu 361
|
| City |
shi jia zhuang |
| State/province |
Hebei |
| ZIP/Postal code |
050017 |
| Country |
China |
| |
|
| Platform ID |
GPL16956 |
| Series (2) |
| GSE77279 |
The long non-coding RNA(lncRNA) expression profiles in human aortic vascular smooth muscle cells (HAVSMCs) |
| GSE77280 |
Long non-coding and circular RNA expression profiles in human aortic vascular smooth muscle cells (HAVSMCs) |
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