|
| Status |
Public on Aug 01, 2012 |
| Title |
normal (grade 1) cartilage, mid zone, rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
donor 77, cartilage, middle zone
|
| Organism |
Homo sapiens |
| Characteristics |
donor: 77
age: 46 yo
gender: Male
tissue: cartilage, middle zone
|
| Treatment protocol |
Osteochondral cores were obtained from the medial and lateral femoral condyles of normal knee joints. The bone portion of each osteochondral core was embedded in paraffin in a standard plastic cassette to allow fixation of the plug in the microtome. The entire cartilage was sliced into 50 μm thick sections from the cartilage surface downwards to the calcified cartilage using a microtome (Microm HM 325, Thermo Scientific, Walldorf, Germany). Each section was transferred to one well of a 96-well plate filled with RNAlater (Qiagen). The sections were segregated into zones for RNA isolation as follows: The upper 10% of zonal slices were allocated to the SZ, the middle 40% were allocated to the MZ and the lower 30% were allocated to the DZ. To avoid overlap between zones, regions of approximately 200 μm between the SZ and MZ and between the MZ and DZ were discarded.
|
| Growth protocol |
Normal human knee joints were procured by tissue banks and processed within 24-60 hours post mortem.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cartilage zones using the Qiagen Rneasy kit with Dnase digestion following the manufacturer's instructions. Total RNA was quantified using NanoDrop (ND-1000). Sample quality was checked with the Agilent 2100 Bioanalyzer using the RNA 6000 Pico LabChip (Agilent Cat. # 5065-4473). Only samples with RIN > 6 were used.
|
| Label |
biotin
|
| Label protocol |
Five nanograms (5 ng) of total RNA was amplified using the NuGEN Ovation Pico WTA System version 1.0. Post amplification, 4ug of the purified cDNA product was processed with the NuGEN WT-Ovation Exon Module version 1.0. Post Exon, 5ug of the purified cDNA product was fragmented and labeled using NuGEN Encore Biotin Module.
|
| |
|
| Hybridization protocol |
5 ug post fragmentation and labeling product was hybridized overnight following the NuGEN Encore Biotin Module protocol. Hybridization and scanning of samples to arrays was performed using the standard NuGEN Hybridization, Cocktail Assembly, and Fluidics Protocols using Affymetrix’s GeneChip Hybridization, Wash, and Stain Kit (Affymetrix P/N 900720), according to NuGEN Encore Biotin Module protocol
|
| Scan protocol |
Chips were scanned using the Affymetrix GeneChip Scanner 3000 7G with default settings and a target intensity of 250 for scaling.
|
| Description |
77M
|
| Data processing |
Data was normalized using both RMA and dCHIP
Log2 RMA signals are presented in the data table below.
Log2 dCHIP signals can be found in a supplementary file linked to the Series record.
The normalized data have filtered out the Affymetrix Control probes and the 30% lowest variant probesets.
|
| |
|
| Submission date |
Aug 01, 2012 |
| Last update date |
Aug 01, 2012 |
| Contact name |
Shawn Patrick Grogan |
| E-mail(s) |
sgrogan@scripps.edu
|
| Phone |
8587848955
|
| Fax |
8587842744
|
| Organization name |
TSRI
|
| Department |
MEM
|
| Lab |
MEM-161
|
| Street address |
10550 N. Torrey Pines Road, MEM161
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (2) |
|
