TY - JOUR
T1 - Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.
AU - McAlinden, Audrey
AU - Haag, Jochen
AU - Bau, Brigitte
AU - Gebhard, Pia M.
AU - Aigner, Thomas
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=16644365690&partnerID=8YFLogxK
U2 - 10.1385/1-59259-810-2:079
DO - 10.1385/1-59259-810-2:079
M3 - Article
C2 - 15280589
AN - SCOPUS:16644365690
SN - 1543-1894
VL - 100
SP - 79
EP - 100
JO - Methods in Molecular Medicine
JF - Methods in Molecular Medicine
ER -