Extraction and isolation of mRNA from adult articular cartilage.
We have developed a method to isolate RNA in high yield from adult articular cartilage. Homogenization of the articular cartilage with a freezer mill, extraction with 4 M guanidinium isothiocyanate/acid-phenol, and ultracentrifugation in cesium trifluoroacetate was found to be an effective and practical method for isolating a high yield of intact RNA from adult canine articular cartilage. The total RNA was suitable for Northern blot analysis. The mRNA that could then be isolated by oligo-dT affinity chromatography was found to be a suitable substrate for in vitro translation, for making a cDNA library, and for PCR amplification.
Adams
M E
ME
Department of Medicine, University of Calgary, Canada.
Huang
D Q
DQ
Yao
L Y
LY
Sandell
L J
LJ
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D002358:Cartilage, Articular / Q000737:chemistry*; D002846:Chromatography, Affinity; D003094:Collagen / Q000235:genetics*; D015342:DNA Probes; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000235:genetics; D014176:Protein Biosynthesis; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D014461:Ultracentrifugation
D053674:Aggrecans; D015342:DNA Probes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011347:Procollagen; D011509:Proteoglycans; D012333:RNA, Messenger; D003094:Collagen