Poly(A)+RNA Isolation
Eukaryotic messenger RNA (mRNA) can be separated from the other RNA species in a total RNA preparation by affinity chromatography by virtue of the presence of a polyadenylic acid “tail,” 20–25 bases long, at the 3′ end of the molecule (1). Oligo(dT)-cellulose is used routinely for homemade affinity columns, but a range of ready-made products is now available from a number of suppliers (e.g., Hybond-mAp, Amersham, Arlington Heights, IL; PolyATract™, Promega, Madison, WI). Oligo(dT)-cellulose consists of a polymer of 10–20 T-residues, covalently linked to a cellulose matrix, which will hybridize to and bind poly(A)+ containing RNA providing the tail is at least 15–20 bases long. Ribosomal and transfer RNA do not bind efficiently to oligo(dT) and can be washed through the column. Bound poly(A)+ RNA can then be eluted with low salt buffers. The protocol given below is a general purpose method designed to give high yields of cDNA synthesis-/in vitro translation-quality mRNA from relatively large amounts of starting material (1–2 mg total RNA).
