HUMAN NORMAL AND OSTEOARTHRITIC KNEE CARTILAGE ISOLATED PATIENTS UNDERGOING KNEE REPLACEMENT SURGERY.
Extracted molecule
total RNA
Extraction protocol
The freshly obtained OA cartilage specimens were washed in sterile PBS and the specimens were immediately snap-frozen at in liquid nitrogen, and subsequently stored at –140 °C until RNA extraction. Normal cartilage were procured from NDRI as frozen samples. The 1-2 grams frozen cartilage were milled into fine powder in liquid nitrogen using Freezer Miller (SPEX, Metuchen, NJ, USA). Total RNA was extracted from pulverized cartilage as described previously (Attur et al., FASEB J. 29, 4107–4121 (2015). The powder cartilage (for one g cartilage /10ml of TRI reagent) was incubated in TRI Reagent (MRC Labs, Cincinnati, OH, USA) for 4 h on a rocker at -4C, and chloroform (10%) 1- 2ml was added and spun at 10,000 rpm for 20min at 4C and the clear aqueous phase was collected in a fresh tube. To the clear aqueous phase, add 0.25 volume of high salt solution [0.8M sodium citrate and 1.2M Nacl (Molecular research center (MRC)] and 0.25 volume of isopropanol per 1ml of Trizol used and mix by inverting several times. Spin immediately at maximum speed for 30min at 4oC.The liquid phase has to be removed carefully and do not disturb the RNA pellet (at this time pellet is very loose). The pellet is dissolved in 100-200ul RNAse few water. The RNA pellet was further cleaned and purified using Qiagen RNeasy mini kit according to the manufacturer’s RNA clean-up protocol (Qiagen, Valencia, CA, and USA). Quality and quantity of RNA were confirmed using a Beckman Spectrophotometer
Label
BIOTIN
Label protocol
Pooling of samples: RNA preparations from a total of 60 different normal and OA donors were used. Five paired sets of RNA preparations of articular cartilage each of five donors in equal quantities were used for analysis. The appropriate pooling of RNA samples had been shown to be statistically valid for microarray experiments. Gene expression profiling was performed with the Affymetrix Human Genome (HG) U133A Arrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. The Genechip array U133A is a comprehensive whole human genome expression array with over 14,500 well characterized transcripts.
Hybridization protocol
Five micrograms of total RNA from pooled RNA samples used for double-stranded (ds) cDNA synthesis by the Gibco BRL SuperScript Choice System (Life Technologies). Purified ds cDNA was used for synthesis of biotin-labeled cRNA with the ENZO BioArray High-yield RNA transcript labeling kit (Affymetrix). The biotinylated cRNA was purified with the RNeasy kit (Qiagen), fragmented at 95°C for 35 min for target preparation. Biotinylated fragmented cRNA probes were hybridized in the microarray U133A (Affymetrix, Santa Clara, CA), which contained probe sets of over 15,000 known transcripts and expressed sequence tags (ESTs). Hybridization was performed at 45°C for 16 h in a hybridization oven (Affymetrix). The microarrays were washed and stained with streptavidin–phycoerythrin conjugate in an Affymetrix Genechip Fluidics Station.
Scan protocol
Fluorescence intensities were scanned with a GeneArray Scanner (Affymetrix) and image analysis was performed with GCOS (Affymetrix). GeneChip Scanner 3000 (Affymetrix) was used for scanning, and image analysis was performed with GCOS (Affymetrix).
Data processing
Data preprocessing, including normalization, modeling, and “presence/ absence” calls, was determined with dChip software (http://biosun1.harvard.edu/complab/dchip). Genes differentially expressed by articular cartilage were identified by comparing gene expression levels between the two groups.
