|
| Status |
Public on Mar 07, 2014 |
| Title |
32_LR_SN |
| Sample type |
RNA |
| |
|
| Source name |
Low responder, stimulated plus natalizumab treatment
|
| Organism |
Homo sapiens |
| Characteristics |
subject: 32
disease status: multiple sclerosis (MS)
gender: male
age: 40
natalizumab responder type: low
tissue: whole blood
cell type: CD4+ T cells
treatment: natalizumab
|
| Treatment protocol |
PBMCs were collected from 8 high responders (HR) and 8 low responders (LR) to natalizumab treatment and were stimulated with anti-CD3/-CD28 antibodies (0.1µg/mL, R&D Systems, Minneapolis, MN, USA), with or without addition of natalizumab (50µg/mL) and cultured for 48h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total CD4+ T cells were enriched by MACS negative sorting (Miltneyi Biotec, Auburn, CA, USA). Total RNA was extracted using TRIreagent (Cincinnati, OH, USA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 15ng total RNA using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, Calif, USA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a total volume of 25ul following the manufacturer's instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4851A) 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 using one color scan setting for 8x60k array slides.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent). Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. 24 samples were analyzed using the GPL16083 Platform, and 8 were analyzed using the GPL16705 Platform. Probes not detectable on either the GPL16083 or GPL16705 Platform were excluded. In addition, only probes common to both Platforms are presented. The raw data was extracted using GeneSpring GX software and was quantile normalized using the limma package in the R program. Batch effects were normalized using ComBat.
|
| |
|
| Submission date |
Mar 07, 2013 |
| Last update date |
Mar 07, 2014 |
| Contact name |
Hui Wang |
| E-mail(s) |
hui.wang@xzhmu.edu.cn
|
| Organization name |
Xuzhou Medical University
|
| Street address |
209 Tongshan Rd
|
| City |
Xuzhou |
| State/province |
Jiangsu |
| ZIP/Postal code |
224001 |
| Country |
China |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE44964 |
Integrated genomic and prospective clinical studies show the importance of modular pleiotropy for disease susceptibility, diagnosis and treatment (dataset 2) |
| GSE44966 |
Integrated genomic and prospective clinical studies show the importance of modular pleiotropy for disease susceptibility, diagnosis and treatment |
|
