Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.
Unlike any other technology in molecular biology, the polymerase chain reaction (PCR) has changed the technological armamentarium of molecular scientists working on cartilage, in terms of outstanding sensitivity and accuracy. Four approaches to determine mRNA expression levels by PCR amplification of specific cDNA sequences are currently in use and are discussed in this chapter: conventional PCR with end-point determination, conventional PCR in the logarithmic amplification phase, conventional PCR using internal competitive DNA fragments, and real-time PCR as offered by TaqMan technology and others. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels within cartilage and cultured chondrocytes, as in other tissues and cell types. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. However, this method cannot account for factors such as efficiency of RNA isolation and reverse transcription conditions. Thus, normalization of the acquired data is required, with all its limitations as described.
Department of Orthopaedic Surgery, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA.
Audrey
A
McAlinden
Jochen
J
Haag
Brigitte
B
Bau
Pia M
PM
Gebhard
Thomas
T
Aigner
D053674:Aggrecans; D002358:Cartilage, Articular / Q000166:cytology*; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics; D015870:Gene Expression; D006609:High Mobility Group Proteins / Q000235:genetics; D006801:Humans; D037181:Lectins, C-Type; D010003:Osteoarthritis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics
D053674:Aggrecans; C499177:COL2A1 protein, human; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors