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High salt SDS-DEP technic for isolation of intact rRNA from Entamoeba histolytica

High salt SDS-DEP technic for isolation of intact rRNA from Entamoeba histolytica

Archivos de Investigacion Medica 13(Supl.3): 23-28

Using a modified SDS-diethylpyrocarbonate (DEP) technique in conjunction with a complex low ionic strength buffer (BTKMS), 3 species of RNA of 4S, 17S and 25S (1:2:1 ratio) were detected in E. histolytica after sucrose gradient centrifugation. The 2:1 ratio of the 17S to 25S RNAs, and the ease with which breakdown of the 25S rRNA occurred under mild conditions attested to the instability of the 25S rRNA. Using an extraction buffer of high salt concentration (0.5 M NaCl) combined with SDS-DEP and relatively low incubation temperature (24 deg C), "undegraded" rRNA (free from RNase) was isolated. Significantly larger yields of 25S rRNA were recovered, i.e. 4S, 17S and 25S RNAs were detected on sucrose gradients in an approximate 1:2:3 ratio. Fractionation by polyacrylamide gel electrophoresis of RNAs isolated with this procedure indicated that the 25S rRNA dissociates to form at least 2 species corresponding to those with S-values of 16S and 17S.

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Accession: 001085059

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PMID: 6295313

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