Removal of ribosomal subunits (and rRNA) from cytoplasmic extracts before solubilization with SDS and deproteinization.
More than 95% of total RNA is composed of ribosomal RNAs (rRNAs) (28S, 18S, 5.8S, and 5S). Here, we present a method that is effective in removing rRNA before extraction and purification of total RNA. If you choose to prepare cytoplasmic RNA and wish to analyze any RNA other than rRNA, it is desirable to eliminate the rRNAs by taking advantage of the fact that the ribosomal subunits are very large (40S and 60S). Few, if any, other cellular RNAs are present in such large macromolecular complexes. The vast majority of rRNAs can be removed by sedimentation. Of course, steps must be taken to avoid co-sedimentation of desired RNAs. Co-sedimentation can be greatly reduced by first dissociating ribosomes into their respective subunits by EDTA treatment. The subunits are then "cleaned" by treatment with high salt and nonionic detergent. Ribosomal subunits remain intact under these conditions and can be sedimented free of other RNAs. Subsequently, the remaining RNAs (messenger RNAs [mRNAs] and all other RNAs) can be purified and analyzed by a variety of methods.
Donald C
DC
Rio
Manuel
M
Ares
Gregory J
GJ
Hannon
Timothy W
TW
Nilsen
D002457:Cell Extracts / Q000737:chemistry*; D002458:Cell Fractionation / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D054657:Ribosome Subunits / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012995:Solubility
D002457:Cell Extracts; D011506:Proteins; D012335:RNA, Ribosomal; D012967:Sodium Dodecyl Sulfate