RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.
An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.
Smale
G
G
Shriners Hospital for Crippled Children, Tampa Unit, Florida.
Sasse
J
J
D000818:Animals; D015152:Blotting, Northern; D002021:Buffers; D002358:Cartilage, Articular / Q000737:chemistry*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D006146:Guanidines / Q000737:chemistry; D011509:Proteoglycans / Q000032:analysis*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D013861:Thiocyanates / Q000737:chemistry; D014269:Trifluoroacetic Acid / Q000737:chemistry*
D002021:Buffers; D006146:Guanidines; D011509:Proteoglycans; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012313:RNA; D014269:Trifluoroacetic Acid