Isolation of Poly(A)+ Messenger RNA Using Magnetic Oligo(dT) Beads

Abstract

This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A)⁺ mRNAs. To elute the poly(A)⁺ mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A)⁺ mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).