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

Cold Spring Harb Protoc. 2019 Oct 1;2019(10). doi: 10.1101/pdb.prot101733.

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).

MeSH terms

  • Cellulose / analogs & derivatives
  • Chromatography, Affinity / methods*
  • DNA, Complementary / genetics
  • Magnetic Phenomena
  • Magnetics*
  • Microspheres*
  • Nucleic Acid Hybridization / methods
  • Oligodeoxyribonucleotides / genetics*
  • Oligodeoxyribonucleotides / metabolism
  • Poly A / genetics*
  • Poly A / isolation & purification
  • Poly A / metabolism
  • RNA / genetics
  • RNA / metabolism
  • RNA, Messenger / genetics*
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism

Substances

  • DNA, Complementary
  • Oligodeoxyribonucleotides
  • RNA, Messenger
  • Poly A
  • RNA
  • Cellulose