QUANTIFICATION OF AGGRECAN AND LINK-PROTEIN MESSENGER-RNA IN HUMAN ARTICULAR-CARTILAGE OF DIFFERENT AGES BY COMPETITIVE REVERSE TRANSCRIPTASE-PCR

A competitive reverse transcriptase-PCR (RT-PCR) assay has been develo ped for the quantification of particular mRNA species in human articul ar cartilage. Competitor RNA species were synthesized that differed fr om the amplified target sequence only by the central insertion of an E coRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be perform ed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. Tile inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumou r necrosis factor in monolayers of human articular chondrocytes quanti fied by this competitive RT-PCR method compared favourably with Northe rn hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related chan ges in aggrecan and link-protein mRNA were therefore quantified in hum an articular cartilage directly after dissection from the joint. The c oncentration of link-protein mRNA was higher in immature cartilage tha n in mature cartilage when expressed relative to the amount of glycera ldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes wer e observed in aggrecan mRNA expression. The ratio of aggrecan to link- protein mRNA was higher in mature cartilage than in immature tissue. T hese age-related differences in the molecular stoichiometry of aggreca n and link-protein mRNA might have implications with respect to the re gulation of the formation and the stability of the proteoglycan aggreg ates in cartilage.