|
| Status |
Public on Feb 17, 2011 |
| Title |
CskKO/mock_biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Csk-/- C57BL/6J MEFs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: Csk-/-
cell type: mouse embryonic fibroblasts (MEFs)
transfection: mock
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Sepazol (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. RNA yield and A260/280 ratio were monitored with a NanoDropND-100 spectrometer (Thermo Scientific, Wilmington, DE), and RNA integrity numbers were assessed with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only RNA extracts with RNA integrity number values >9 were included in further analyses.
|
| Label |
Cy3
|
| Label protocol |
One hundred nanograms of aliquots of total RNA was used for making miRNA probes according to the Agilent protocol (ver. 1.6). Briefly, total RNA was dephosphorylated with calf intestine alkaline phosphatase (Takara Bio, Shiga, Japan), denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase (Takara Bio, Shiga, Japan) using the miRNA Labeling Reagent and Hybridization Kit (5190-0408, Agilent).
|
| |
|
| Hybridization protocol |
Refer to Agilent miRNA hybridisation protocol. Probes were hybridized at 55°C for 20 hours with rotation. Then the slides were washed by Gene expression wash buffer 1 at room temperature for 5 minutes and by Gene expression wash buffer 2 at 37°C for 5 minutes.
|
| Scan protocol |
After hybridization and washing, the slides were scanned using an Agilent scanner (G2505B). Images were extracted using Agilent Feature Extraction software (ver. 9.5.3.1) and Agilent GeneSpring GX software (ver. 10.0.2).
|
| Description |
CskKO/mock-rep2
Csk-/- mouse embryonic fibroblasts (Csk-/- MEFs).
|
| Data processing |
Data were processed by the Agilent GeneSpring GX software. Total gene signal from GeneView data files extracted with default settings in Agilent Feature Extraction, which summarized the measurements of all probes for each gene on an array, was used as signal for miRNAs. To avoid missing values after log-transformation, any signal which was still less than 1 was reset to be equal to 1. Differences in miRNA expressions between the 4 pairs was determinate if the fold change of expression values was >2.0 and the p value was <0.05 using Student's t test for further analysis.
|
| |
|
| Submission date |
Aug 04, 2010 |
| Last update date |
Feb 18, 2011 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9756 |
| Series (1) |
| GSE23426 |
Analysis of the miRNA profile associated with c-Src-mediated transformation |
|
