A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation.
High concentrations of KI were found to efficiently protect RNA against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72,000 x g for 24 h. The RNA pellets were found to be readily dissolved in bidistilled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus RNA was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecular-weight mRNA.
Münzner
P
P
Institut für Allgemeine Botanik und Botanischer Garten, Universität Hamburg, F.R.G.
Voigt
J
J
D015152:Blotting, Northern; D002499:Centrifugation, Density Gradient / Q000379:methods; D000460:Chlorophyta / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D008970:Molecular Weight; D011061:Poly A / Q000302:isolation & purification; D011193:Potassium Iodide; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*
D012333:RNA, Messenger; D011193:Potassium Iodide; D011061:Poly A; D012313:RNA