Intervertebral disc tissue was harvested from spinal segments from levels T12-L1 to L4-L5; NP and AF regions were dissected and separated. Cells were isolated from the tissues using sequential pronase and type II collagenase digestion.
Extracted molecule
total RNA
Extraction protocol
Cells were lysed in TRI Reagent (Molecular Research Center, Cincinnati, OH) and RNA was isolated using a modified TriSpin method. Bromo-chloro-propane was added to the lysate, phases were separated, and ethanol added to the aqueous phase. Total RNA was extracted using the SV Total RNA Isolation System (Promega), which includes an on-column DNAse digestion, and eluted in RNase-free water.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip 3000 7G scanner.
Description
172A
Data processing
R/Bioconductor gcrma preprocessing using remapped probesets (CDF:HGU133Plus2_Hs_ENTREZG, version 16.0.0). Batch correction via combat in the bioconductor sva package. Statistical analysis using limma package.
