|
| Status |
Public on May 21, 2012 |
| Title |
293T-mock |
| Sample type |
RNA |
| |
|
| Source name |
nonhepatic cells
|
| Organism |
Homo sapiens |
| Characteristics |
infection status: Mock-treatment
cell line: 293T
|
| Treatment protocol |
Infected cell (HCV+): By using lentiviral vector, microRNA 122 was expressed in the cells. The cells were infected with cell cultured HCV (HCVcc) at an MOI of 10. RNA was collected 24 hours post-infection.
|
| Growth protocol |
Cells were cultured at 37 °C under the condition of a humidified atmosphere and 5% CO2 and maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted from cancer cell lines in RNA later (Ambion), derived from Hep3B, Huh7, Hec1B and 293T using miRNeasy extraction kit (Qiagen) following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNA was labeled using Low Input Quick-Amp Labeling Kit (Agilent Technologies, Pato Alto, CA, USA). In brief, cDNA was reverse transcribed from 100 ng of total RNA with an oligo-dT-T7 promoter primer and AffinityScript MT -RT. Fluorescent antisense cRNA was synthesized with cyanine 3-CTP (Cy3-CTP), and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with miRNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Before hybridization, 1650 ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual, and scanning of microarrays was performed with 5µm resolution using a DNA microarray laser scanner (Agilent Technologies).
|
| Scan protocol |
The array was scanned using Agilent G2505C DNA microarray scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
|
| Description |
mock-treated 293T cells
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction, LOWESS normalization, and dye-normalization .
|
| |
|
| Submission date |
Oct 11, 2011 |
| Last update date |
May 21, 2012 |
| 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 |
GPL10332 |
| Series (2) |
| GSE32886 |
Hepatitis C virus-induced expression profiling of Hepatic and non-hepatic cancer cell lines [mock-treated] |
| GSE70781 |
Hepatitis C virus-induced expression profiling of Hepatic and non-hepatic cancer cell lines |
|
