Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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

(Protocol summary only for purposes of this preview site)

The following protocol, adapted from Jakobsen et al. (1994), provides 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 65C70C, 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). Background information on the isolation of poly(A) mRNA is contained in the box Poly(A) mRNA.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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