mRNA Synthesis Protocol with Modified Nucleotides using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080)

mRNAs containing modified nucleotides (such as 5mCTP and Pseudo-UTP) have been shown to suppress RNA-mediated innate immune activation in vivo.  The HiScribe T7 mRNA Kit with CleanCap Reagent AG is formulated to allow for complete substitution of unlabeled NTPs with a modified version of the NTP.  Inclusion of most modified NTPs (not included) in the in vitro transcription reaction will not affect final RNA yield, however some modified NTPs may impact transcription efficiency.  

The reaction conditions in the example below demonstrates a complete substitution of Pseudo-UTP for UTP.  If another modified NTP is being used, swap in the modified version for the unmodified version at equimolar concentration in the final reaction as the stock concentration of modified NTPs may not be the same as the unmodified version provided in the kit.

We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination.  Reactions are typically 20 µl but can be scaled as needed.

  1. Thaw the necessary kit components to room temperature (Reaction buffer, NTPs, modified NTP, CleanCap Reagent AG and Template DNA), mix and microfuge briefly to collect solutions to the bottom of the tubes.  Keep the Enzyme Mix on ice.

  2. If several reactions will be performed a master mix of Reaction Buffer, NTPs and CleanCap can be prepared according to the volumes per reaction below.  Use 12 µl of the Master Mix per reaction.

  3. Assemble the reaction at room temperature in the following order:

    Nuclease-free water X µl
    10X T7 CleanCap Reagent AG Reaction Buffer
    2 µl
    ATP (60 mM)
    2 µl
    6 mM final
    Pseudo-UTP (50 mM)
    2 µl
    5 mM final
    CTP (50 mM)
    2 µl
    5 mM final
    GTP (50 mM)
    2 µl
    5 mM final
    Cap Analog (40 mM)
    2 µl
    4 mM final
    Template DNA
    X µl
    1 µg
    DTT (0.1M)*
    1 µl
    5 mM
    T7 RNA Polymerase Mix
    2 µl
    Total Reaction Volume
    20 µl

    *Addition of DTT to the reaction is optional but recommended. See Troubleshooting section for details.

  4. Gently mix the reaction by pipetting up and down and microfuge briefly.  Incubate at 37°C for 2 hours.

    For reaction times of 60 minutes or less, a water bath or heat block may be used.  For reactions longer than 60 minutes, we recommend using a dry air incubator or PCR instrument set to 37 C (with a heated lid) to prevent evaporation. For reactions with transcripts < 0.3 kb incubations up to 16 hours or overnight can be performed.

  5. Optional: Bring the reaction volume up to 50 µl with nuclease-free water.  Add 2 µl of DNase I, mix well and incubate at 37°C for 15 minutes.

  6. Proceed with mRNA purification.