mRNA Fragmentation Protocol (E6110)
Overview
Starting Material: Purified mRNA (50–250 ng)
Protocol
- mRNA Fragmentation Protocol
- 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
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Total volume: 20 μl - Incubate in a preheated thermal cycler for 5 minutes at 94°C. This is the optimal condition for eukaryotic mRNA (see Figure 1 on the product page). Other types of mRNA may require optimizing incubation time to obtain desired fragment size distribution.
- Transfer tube to ice.
- Add 2 μl 10X RNA Fragmentation Stop Solution.
- Mix the following components in a sterile PCR tube:
- Clean Up Fragmented RNA Using RNeasy MinElute Spin Columns
- Add 78 μl of the Nuclease-Free Water to the 22 μl fragmented RNA from step 4. Purify sample using RNeasy MinElute Cleanup Kit (Qiagen #74204) following manufacture instructions. Elute in 15.5 μl Nuclease-Free Water. The recovered volume should be ~14.5 μl.
Note: column purification removes short RNA Fragments and enriches
the sample for RNA fragments longer than 200 nucleotides.
- Add 78 μl of the Nuclease-Free Water to the 22 μl fragmented RNA from step 4. Purify sample using RNeasy MinElute Cleanup Kit (Qiagen #74204) following manufacture instructions. Elute in 15.5 μl Nuclease-Free Water. The recovered volume should be ~14.5 μl.
- Alternatively, Clean Up Fragmented RNA Using Ethanol Precipitation
- Mix the following components in a sterile 1.5 ml microcentrifuge tube:
Fragmented RNA from Step 4: 22 μl
3M Sodium Acetate, pH 5.2: 2 μl
Linear Acrylamide, 10 mg/ml: 1–2 μl
100% Ethanol: 60 μl
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Total volume: 85–86 μl - Incubate at -80°C for 30 minutes.
- Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.
- Carefully remove ethanol.
- Wash pellet with 300 μl of 70% ethanol.
- Centrifuge and carefully remove 70% ethanol.
- Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
- Resuspend in 14.5 μl Nuclease-Free Water.
- Mix the following components in a sterile 1.5 ml microcentrifuge tube: