Home Protocols mRNA Isolation using Streptavidin Magnetic Beads (NEB #S1420 and NEB #S1421)

mRNA Isolation using Streptavidin Magnetic Beads (NEB #S1420 and NEB #S1421)


For use with Streptavidin Magnetic Beads (S1420) and Hydrophilic Streptavidin Magnetic Beads (S1421).

Introduction

mRNA Isolation using Streptavidin Magnetic Beads: For the isolation of mRNA from 100 µg of total RNA or 5 x 106 cells. The yield of poly(A)+ RNA will vary with the type of tissue or cells used.

Protocol

  1. Prepare a 65°C bath.
  2. Prewarm Elution Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA] in 70°C bath.
  3. Place Low Salt Buffer in ice bath.
  4. Dissolve 1.0 A260 unit of biotin-(dT)18 in 500 µl of Wash/Binding Buffer [0.5 M NaCl, 20 mM Tris-HCl(pH 7.5), 1 mM EDTA]. Final concentration 8 pmol/µl.
  5. Aliquot 125 µl (500 µg) of Streptavidin Magnetic Beads per 100 µg of total RNA into a clean RNase-free microcentrifuge tube. Add 100 µl of Wash/Binding Buffer and vortex to suspend beads. Apply magnet to side of tube for approximately 30 seconds. Remove and discard supernatant.
  6. Add 25 µl of biotin-(dT)18 solution to magnetic beads and vortex to suspend beads. Incubate at room temperature for 5 minutes with occasional agitation by hand. Apply magnet then remove and discard supernatant.
  7. Wash beads by adding 100 µl of Wash/Binding Buffer. Vortex to suspend then apply magnet and discard supernatant. Repeat wash.
  8. Dissolve 100 µg of total RNA in 50 µl of Wash/Binding Buffer and heat at 65°C for 5 minutes Then quickly chill in an ice bath for 3 minutes.
  9. Add total RNA sample to previously prepared magnetic beads. Vortex to suspend the particles then incubate at room temperature for 10 minutes with occasional agitation by hand.
  10. Apply magnet then remove supernatant. Add 100 µl of Wash/Binding Buffer, vortex to suspend beads. Apply magnet then remove and discard supernatant. Repeat washing with fresh Wash Buffer.
  11. Add 100 µl of cold Low Salt Buffer [0.15 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA] to beads, vortex to suspend. Apply magnet then remove and discard supernatant.
  12. Add 25 µl of prewarmed Elution Buffer, vortex to suspend beads then incubate at room temperature for 2 minutes.
  13. Apply magnet then transfer supernatant to a clean RNase-free microcentrifuge tube.
  14. Repeat elution with 25 µl of fresh Elution Buffer. Apply magnet and add supernatant to first mRNA elution. At this point quantification of isolated poly(A)+ can be done by spectrophotometric measurement (1 A260 = approximately 40 µg) or simply proceed to reverse transcription reaction.