RNA extraction from cartilage.
The direct isolation of RNA from cartilage has often proved difficult owing to a number of factors. Cartilage has a low cell content and contains an extracellular matrix rich in proteoglycans, which copurify with the RNA as they are large and negatively charged macromolecules. In our laboratory, we are interested in searching for genes differentially expressed in chondrocytes in diverse in vivo situations, for instance during maturation of chondrocytes in the growth plate or during cartilage degeneration. We found that treatment by proteinase K in 1 M guanidinium isothiocyanate prior to cesium trifluoroacetate ultracentrifugation was crucial to increase the yield and purity of RNA extracted from cartilage matrix. This protocol indeed led to reproducible patterns of differential display reverse transcriptase-polymerase chain reaction (RT-PCR) and should be useful for identifying genes differentially expressed by chondrocytes in situ.
Mallein-Gerin
Frédéric
F
Laboratory of Biology and Engineering of Cartilege, CNRS UMR 5086, Institut de Biologie et Chimie des Protéines, Lyon, France.
Gouttenoire
Jérôme
J
D000818:Animals; D002356:Cartilage / Q000737:chemistry*; D005821:Genetic Techniques; D006801:Humans; D012313:RNA / Q000302:isolation & purification*
D012313:RNA