|
| Status |
Public on May 15, 2020 |
| Title |
Arthritic mouse 2 |
| Sample type |
RNA |
| |
|
| Source name |
Spleen
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
tissue: spleen
cell type: B cell
gender: female
|
| Treatment protocol |
BALB/c mice were immunized with human PG three times, and after the third treatment B cells were affinity purified from spleens collected from the sacrificed mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Direct-Zol™ RNA MiniPrep Plus kit (Zymo Research, Irvine, CA, USA) treated with Turbo™ DNase (ThermoFisher Scientific, Waltham, MA, USA) and purified with RNA Clean and Concentrator™-5 kit (Zymo).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS 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.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 10 days of 3rd PG-injection
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 15, 2017 |
| Last update date |
May 15, 2020 |
| Contact name |
Tibor T Glant |
| E-mail(s) |
Tibor_Glant@rush.edu
|
| Organization name |
Rush University Medical Center
|
| Department |
Orthopedic Surgery
|
| Lab |
Molecular Medicine
|
| Street address |
1735 West Harrison
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60612 |
| Country |
USA |
| |
|
| Platform ID |
GPL15691 |
| Series (1) |
| GSE98932 |
Autoimmune arthritis-associated gene expression in B cells isolated from mice with proteoglycan (PG)-induced arthritis (PGIA) |
|
