|
| Status |
Public on Dec 05, 2012 |
| Title |
TGFbeta MCT 3 |
| Sample type |
RNA |
| |
|
| Source name |
MCT_TGFbeta
|
| Organism |
Mus musculus |
| Characteristics |
strain background: SJL H-2S
cell type: Mouse renal tubular cells (MCT)
stimulated with: GFbeta 3ng/ml for 72 hrs
molecule subtype: miRNA
|
| Treatment protocol |
Prior to TGF-β stimulation, the medium was changed to DMEM supplemented with 0.5% FCS, and TGF-β was subsequently added to the medium at 3 ng/ml on the next day. Cells were treated with TGF-βfor three days.
|
| Growth protocol |
A mouse renal tubular cell line, MCT was maintained in Dulbecco's modified Eagles's medium (DMEM) and 10% fetal calf serum (FCS). Prior to TGF-β stimulation, the medium was changed to DMEM supplemented with 0.5% FCS, and TGF-β was subsequently added to the medium at 3 ng/ml on the next day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.And RNA was prepared using the , miRNAeasy mini (QIAGEN, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
pCp Cyanine-3 (Cy3) labeled miRNA was prepared from 100ng total RNA using the miRNA Complete Labeling Kit(Agilent) according to the manufacturer's instructions.After labering reaction,completely dray the samples.Use a vacuum concentrator at the midium-high heat setting.. Resuspend the dried sample in 18ul of nuclease-free water.
|
| |
|
| Hybridization protocol |
18ul of Cy3-labelled miRNA was fragmented following the manufacturers instructions. On completion of the fragmentation reaction, 4.5ul of 10x GE Block and 22.5ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse miRNA Microarray Kit (V2), 8x15K (Based on Sanger miRBase release 12.0)ver2(G4471A) for 20 hours at 55℃,20RPM in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute at 37℃ GE Wash buffer 2 (Agilent), then dried immediately by air spray
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 5% ).
|
| Description |
SAMPLE 6
|
| Data processing |
The scanned images were analyzed with Feature Extraction software Ver 10.1.1(Agilent).
|
| |
|
| Submission date |
Dec 04, 2012 |
| Last update date |
Dec 05, 2012 |
| Contact name |
Ryuji Morizane |
| E-mail(s) |
[email protected]
|
| Organization name |
Keio University School of Medicine
|
| Department |
Internal Medicine
|
| Lab |
Renal
|
| Street address |
35 shinanomachi, shinjukuku
|
| City |
Tokyo |
| ZIP/Postal code |
1608582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10384 |
| Series (2) |
| GSE42718 |
miRNA microarray of mouse renal tubular cell with or without TGFbeta |
| GSE42719 |
miR-34c attenuates epithelial-mesenchymal transition and renal fibrosis of kidneys with ureteral obstruction. |
|
