NEBNext® RNase III RNA Fragmentation Module (E6146)

Protocol

1. Starting Material: Purified mRNA (50–250 nanograms)
   1. Mix the following components in a sterile PCR tube:

      	Volume (μl)
      Purified mRNA (100 nanograms)	variable
      RNase III (1 unit/μl)	1
      RNase III Reaction Buffer (10X)	2
      Nuclease-Free Water	variable
      Total volume	20

   2. Incubate in a preheated thermal cycler for 5 minutes at 37°C.*
   3. Add 80 μl of cold Nuclease-Free water.
   4. Transfer tube to ice.
   5. Clean up RNA fragments using a RNA column purification kit or ethanol precipitation.
      
      * These conditions have been optimized to fragment 100 nanograms of mammalian mRNA into RNA fragments with a normal distribution with a mean peak at 200 nucleotides (see figure 1 on the product page). Other types of RNA (Plant, Bacterial, Yeast) or other amounts of mammalian mRNA might require adjusting the amount of enzyme and/or incubation time depending on the RNA fragment size desired.
2. Clean Up Fragmented RNA Using RNeasy MinElute Spin Columns
   1. Purify sample using RNeasy MinElute Cleanup Kit (Qiagen #74204) following the manufacturer’s instructions. Elute with 14 μl nuclease-free water. The recovered volume should be ~12 μl. 
      
      Note: column purification removes short RNA Fragments and enriches
      the sample for RNA fragments longer than 200 nucleotides.
3. Alternatively, Clean Up Fragmented RNA Using Ethanol Precipitation
   1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:

      	Volume (μl)
      Fragmented RNA from Step 4	100
      3M Sodium Acetate, pH 5.2	10
      Linear Acrylamide, 10 mg/ml	1–2
      100% Ethanol	300
      Total volume	411–412

   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% freshly prepared 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 appropriate volume of Nuclease-Free Water.
4. Assess the Yield and the Size Distribution of the Purified RNA Fragments.
   
   Run 1 μl of the purified RNA fragments in the Agilent Bioanalyzer 2100 using a RNA Pico chip (see figure 1 on the product page).