Isolation and analysis of ribonucleic acids from skeletal tissues.
We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.
Nemeth
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Orthopaedic Research Unit, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Heydemann
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Bolander
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D000818:Animals; D001842:Bone and Bones / Q000032:analysis*; D002146:Bony Callus / Q000032:analysis; D002356:Cartilage / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D012313:RNA