|
| Status |
Public on Sep 11, 2015 |
| Title |
ES-CM-siScr-2 |
| Sample type |
RNA |
| |
|
| Source name |
embryonic stem cells-derived cardiomyocytes treated with si-Scramble
|
| Organism |
Mus musculus |
| Characteristics |
cell line: CGR8
transfection: siRNA-Scramble
|
| Treatment protocol |
The cells were transfected with siScr or siErbB4 for 48 hours using Pepmute transfection agent
|
| Growth protocol |
Mouse embryonic stem cells-derived cardiomyocytes were cultured in DMEM-LG+2%FBS. The cultures were incubated for 6 or 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA were extracted using RNEasy Plus Mini Kit with DNAse treatment
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) 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 |
1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse GE 4*44K v2 Microarrays (G4846A) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) at 535 nm for Cy3.
|
| Description |
Gene expression after transfected with si-Scr
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep0 and Grid: 026655_D_F_20130207) to obtain background subtracted and spatially detrended Processed Signal intensities.
GeneSpring GX v12.6 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Follow by Percentile Shift Normalization.
|
| |
|
| Submission date |
Jun 15, 2015 |
| Last update date |
Sep 11, 2015 |
| Contact name |
Desy S Lee |
| E-mail(s) |
kh4ishi@gmail.com
|
| Organization name |
NCKU
|
| Street address |
University Road
|
| City |
Tainan |
| ZIP/Postal code |
701 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE69897 |
Gene expression profile of mouse embryonic stem cells derived cardiomyocytes following the knockdown of ErbB4 |
|
