RNA extraction from Cartilage from RLV
Approximately 2µg of fresh human articular cartilage was diced, washed briefly with PBS pH 7.4, blotted dry and ten snap frozen in liquid nitrogen. The cartilage was crushed using either a pestle and mortar, ensuring that the tissue was covered by liquid nitrogen through out, or a dismembrator (B. Braun Biotech. Int.) for 1 minute at 1800rpm. The crushed cartilage was transferred into a tube containing 4ml Tri reagent/gram tissue (Tri reagent contains guanidine isothiocyanate to lyse the cells and phenol to extract the RNA) and placed on a rotator for 30 minutes. Samples were centrifuged at 3,000g for 15 minutes to remove cell debris and the supernatant collected. One ml of supernatant was layered onto 900µl of 1.60g/ml cesium trifluoroacetate (CsTFA) (Pharmacia-BioTech) in Beckman polyallomer ultracentrifuge tubes (11x34mm). Samples were centrifuged using a Beckman TLS55 rotor in a model TL100 tabletop ultracentrifuge at 55,000 rpm (~200,000g) for 3 hours at 18 degees Centigrade. After removing the supernatant, the RNA pellet in each tube was dissolved in 100µl water, and the pellets from 4 tubes were transferred in to a 1.5ml eppendorf tube. After the addition of 20µl of 5M NaCl and 2.5 volumes (1ml) of absolute ethanol, samples were incubated at -20 degrees overnight and centrifuged at 12,000g for 15 minutes. After removing the supernatant, RNA pellets were washed with 75% ethanol, allowed to air dry, dissolved in 75µl water, and stored at -20 prior ot analysis.