Analysis of Human Osteoarthritic Connective Tissue by Laser Capture Microdissection and QRT-PCR

Abstract

Gene expression levels for type II collagen and aggrecan have been determined as potential measures and disease markers of human osteoarthritis in patients undergoing total knee arthroplasty. In this regard, specimens of affected articular cartilage obtained intraoperatively at the time of surgery were placed in RNAlater^TM to maintain RNA integrity and subsequently frozen-sectioned. Individual or small numbers of chondrocytes were isolated by laser capture microdissection and their total RNA was extracted and analyzed by quantitative reverse transcription-polymerase chain reaction. Results indicate that type II collagen and aggrecan mRNA expression from specific cells in osteoarthritic tissues are detectable and reproducible using these approaches. Our work is the first to demonstrate successful isolation of RNA limited to chondrocytes comprising small quantities of human osteoarthritic material. The study presents a new avenue by which the disease and its progression may be critically assayed.

Keywords:

• Aggrecan Gene Expression
• Cartilage
• Human Osteoarthritis
• Laser Capture Microdissection
• QRT-PCR
• Type II Collagen