|
| Status |
Public on Jan 04, 2017 |
| Title |
tumor-free mice treated with anti-IL-6R Ab #6 |
| Sample type |
RNA |
| |
|
| Source name |
isolated CD4 T cells #6
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
host: tumor-free mice
treatment: anti-IL-6R Ab
cell type: CD4+ T cell
|
| Treatment protocol |
C57BL/6 mice were inculated with ovalbumin(OVA)-expressing MCA205 fibrosarcoma, and then were transferred with OVA-specific TCR transgenic CD4+ T cells. Furthermore, those mice were immunized by the transfer of OVA peptide-pulsed dendritic cells with or without simultaneous injection of anti-IL-6R Ab. The same treatments were also conducted in tumor-free mice. Four days after in vivo immunization, CD4+ T cells were isolated from those mice (four groups). The total RNA isolated from those CD4+ T cells were utilized for microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from CD4+ T cells using the TRIZOL Reagent (Thermo Fisher Scientific), followed by the further purification via RNeasy mini columes (QIAGEN). 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 |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the Low Input Quick Amp Labeling Kit and Agilent RNA Spike-In 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.
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| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >14.0 pmol Cy3/ug cDNA) was fragmented at 60 °C for 30 minutes, and were hybridized to SurePrint G3 Mouse GE microarray 8X60K v2 Microarrays Kit (G4853B) using Gene Expression Hybridization Kit (Agilent) 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 after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in CD4+ T cells from tumor-free, anti-IL-6R Ab-treated mice
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software Version 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 074809_D_F_20150624) to obtain background subtracted and spatially detrended Processed Signal intensities. Raw fluorescent data was extracted using GeneSpring GX software 14.5.0 (Agilent), were then subjected to log2 transformation and 75th percentile normalization for analysis.
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| |
|
| Submission date |
Jan 04, 2017 |
| Last update date |
Jan 04, 2017 |
| Contact name |
Hirotake Tsukamoto |
| E-mail(s) |
[email protected]
|
| Phone |
+81753667439
|
| Organization name |
Kyoto University
|
| Department |
Center for Cancer Immunotherapy and Immunobiology
|
| Lab |
Division of Clinical Immunology and Cancer Immunotherapy
|
| Street address |
53 Kawahara-cho Shogoin
|
| City |
Kyoto |
| State/province |
Sakyo-Ku |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE93105 |
Investigation of molecular events underlying the IL-6-mediated Th1 inhibition in tumor-bearing mice |
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