Quantifying Gene Expression in OA Tissues

The homogenized tissue was placed on ice, and 100mg was weighed and separated for RNA isolation using a kit (Versagene’s 5-Prime RNA Isolation Kit⁴). RNAse free procedures and filtered pipettes were used throughout the isolation procedure. The nanodrop spectrophotometer (ND-1000 Spectrophotometer⁵) was used to analyze the amount and purity of the RNA product. Concentrations ranged from 3.8–51.8ng/ul, and one joint (mild OA) was excluded as its RNA concentration was less than 3.0ng/ul and its 260/280 wavelength ratio was <1.6.
Gene expression was quantified through the use of quantitative real-time PCR (ABI PRISM 7900 Sequence Detection System ⁶). Primers and labeled probes (6-FAM as the 5′ reporter label and TAMRA as the 3′ quenching label) were created using commercial software (ABI Primer Express Software 2.0b8a⁶). Sequences for these primers and probes were generated from Genbank⁷ or available from clones within the laboratory of Dr Alan Nixon. Copy number was quantified for the mRNA of TNF-α, IL-1β, aggrecanase 1 (ADAMTS-4), aggrecanase 2 (ADAMTS-5), MMP-13, and IL-17 in both the synovium and the cartilage. Collagen type I alpha 1(Col-1) was quantified in synovium, and collagen type IIB (Col-2B) and aggrecan were quantified in cartilage. The housekeeping gene used was 18S, and the 18S copy number was similar for all tissues used. Data used for statistical analysis was copy number per microgram.