Author:
Green Michael R.,Sambrook Joseph
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).
Publisher
Cold Spring Harbor Laboratory
Subject
General Biochemistry, Genetics and Molecular Biology
Cited by
18 articles.
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