|
| Status |
Public on Mar 30, 2012 |
| Title |
BMP2(-),LOXL2(+) |
| Sample type |
RNA |
| |
|
| Source name |
primary osteoblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: pre-osteoblasts
treatment: BMP2(-),LOXL2(+)
|
| Treatment protocol |
Primary osteoblasts were treated with BMP2 (200 ng/ml) and or LOXL2 (4 μg/ml) for 72h.
|
| Growth protocol |
Primary osteoblasts were harvested and cultured in the presence of 10% FBS for culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Rneasy mini kit (Qiagen) extraction of total RNA was performed according to the manufacture's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low RNA Input Liner Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 50 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 ul of Agilent 2x HI-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarrays (G4852A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (20 bit, Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100% ).
|
| Description |
Osteoblastgenesis
US11060402_252800512938_S01_GE1_107_Sep09_1_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028005_D_F_20110722) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Mar 29, 2012 |
| Last update date |
Mar 30, 2012 |
| Contact name |
Dai Suzuki |
| E-mail(s) |
dai.suzuki@dent.showa-u.ac.jp
|
| Phone |
81-3-3784-8163
|
| Fax |
81-3-3784-5555
|
| Organization name |
Showa University
|
| Department |
Biochemistry
|
| Street address |
1-5-8 Hatanodai, Shinagawa
|
| City |
Tokyo |
| ZIP/Postal code |
142-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE36925 |
Mouse primary osteoblasts treated with/without BMP2 |
|
