mRNA Fragmentation Protocol (E6114)

Introduction

Starting Material: mRNA purified from 1–10 μg total RNA

Protocol

1. mRNA Fragmentation Protocol
   1. Mix the following components in a sterile PCR tube:
      Purified mRNA: 1–18 μl 
      10X RNA Fragmentation Reaction Buffer:  2 μl 
      Nuclease-Free Water: variable 
      ------------------------------------------------------
      Total volume: 20 μl
   2. Incubate in a preheated thermal cycler for exactly 5 minutes at 94°C.
   3. Transfer tube to ice.
   4. Add 2 μl 10X RNA Fragmentation Stop Solution.
2. Ethanol Precipitation of Fragmented mRNA
   1. Mix the following components in a sterile 1.5 ml microcentrifuge tube: 
      Fragmented mRNA: 22 μl 
      3 M Sodium Acetate, pH 5.2:  2 μl 
      Linear Acrylamide, 10 mg/ml: 1–2 μl 
      100% Ethanol: 60 μl 
      ------------------------------------------------------
      Total volume: 85–86 μl
   2. Incubate at -80°C for 30 minutes.
   3. Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.
   4. Carefully remove ethanol.
   5. Wash pellet with 300 μl of 70% ethanol.
   6. Centrifuge and carefully remove 70% ethanol.
   7. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
   8. Resuspend in 13.5 μl Nuclease-Free Water.
3. Alternative Protocol:
   Clean up Fragmented RNA using a RNA column purification kit such as Qiagen’s RNeasy Minelute Kit, eluting the RNA in 13.5 μl Nuclease-Free Water or elution buffer.