In vitro transcription, capping, and 2'-O methylation of long RNAs

Materials

  • 10X transcription buffer
    • 300mM Tris-Cl pH (RT) = 8.1
    • 250mM MgCl2
    • 0.1% Triton X-100
    • 20mM spermidine
    • 100mM DTT
  • NEB Vaccinia capping enzyme system (M2080S)
    • NEB 2'-O-methyltransferase (M0366S)
      • T7 polymerase (1mg/mL; purified separately)
        • Ambion NorthernMAX glyoxal loading dye (AM8551)
          • Ambion GlycoBlue co-precipitant (AM9516)
            • 0.3M NaOAc pH 5.2
              • 50mM EDTA
                • RQ1 RNase-free DNase
                  • Zymo Clean & Concentrator 25 columns
                    • One of these two RNA ladders:
                      • https://www.neb.com/products/n0362-ssrna-ladder (for transcripts > 2kb)
                      • https://www.thermofisher.com/order/catalog/product/15623100 (for transcripts < 2kb)

                    PCR amplification of template

                    1. Primers:

                    pA60 txn rev TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTG CAG

                    pA60 txn fwd CGG CCA GTG AAT TCG AGC TCT AAT ACG ACT CAC TAT AGG

                    1. Reactions (4x 100µL for each one): Master mix (10*4 -> make 45 rxns worth)
                    Make 4 x 100 ul reactions for each template
                    ReagentAmount per 100 ul rxn
                    HF buffer20 ul
                    dNTP2 ul
                    fwd primer (100 uM)0.5 ul
                    rev primer (100 uM) 0.5 ul
                    phusion polymerase1 ul
                    template (50 ng total)10 ul
                    water66 ul
                    1. Run PCR with a two step protocol:

                    72 degrees annealing/extension for 3 minutes

                    98 degrees melting for 30 seconds

                    repeast 30 cycles

                    10 minute final extension time

                    1. Load all of the PCRs into an agarose gel and verify then size of the template band and then gel purify it

                    In vitro transcription of RNA

                    1. Warm all reagents except T7 polymerase and Superase:IN to room temperature and assemble the reaction at RT. Adding cold spermidine to template can cause precipitation of the template.
                    1. Mix the following together in a 1.5mL tube
                    Reagent100 ul reaction (ul each)
                    Template1 to 5 micrograms worth
                    ACGU (25 mM) 30
                    10X txn buffer (RT)10
                    DTT (1M)2
                    MgCl2 (1M) 1
                    T7 polymerase10
                    Superase:IN1
                    DEPC H2OTo 100 ul
                    1. Incubate at 37 degrees C in a stable incubator - a water bath or stable dry incubator. Warm rooms and heat blocks can have higher variability in temperature.
                    1. Wait 1 hour and check the reactions - they should be somewhat cloudy. If they are, add 1µL of 1mg/mL pyrophosphatase to each (Roche 10108987001)
                      1 h
                    1. Allow the reactions to proceed for 3-4 hours.
                      3 h
                    1. Add 1µL of RQ1 RNase-free DNase to each reaction and incubate for 30 minutes.
                      30 m
                    1. Precipitate by adding 200µL 0.3M NaOAc pH 5.2, 1µL GlycoBlue and 750µL 100% EtOH.
                    1. PAUSEIncubate at -80 for >1 hour to overnight.

                    Resuspending EtOH precipitated RNA

                    1. Pellet precipitations by centrifugation at 16,000g at 4 degrees for 25 minutes.
                      25 m
                    1. Aspirate supernatant and discard
                    1. Wash with 500µL -20 degree 70% EtOH
                    1. Centrifuge at 16,000g and 4 degrees for 5 minutes
                    1. Aspirate supernatant and discard
                    1. Quick-spin and remove residual ethanol with a P20 pipet
                    1. Allow pellet to air dry for 5 minutes; do not overdry
                    1. Resuspend pellet in 100µL 50mM EDTA
                    1. Purify RNA over Zymo RNA Clean & Concentrator 25 column following small RNA removal protocol and elute in 30µL DEPC H2O
                    1. Recommended but optional: assess RNA integrity by running a Glyoxal gel before proceeding
                    1. Measure concentration of RNA on nanodrop

                    Capping and 2'-O-Methylation

                    1. Dilute 20µg of RNA in 26µL DEPC H2O
                    1. Prepare a 4mM stock of SAM from the supplied 32mM stock (4µL 32mM and 28µL DEPC H2O for up to 15 reactions)
                    1. Assemble a master mix according to the following recipe:
                    Reagentper 40 ul reaction
                    10X capping buffer4
                    10 mM GTP2
                    4 mM SAM2
                    Vaccinia capping enzyme2
                    2'-O-Me transferase2
                    RNasin (promega)2
                    1. Add the RNA and water to each tube and add 14µL of the master mix to each
                    1. Incubate at 37 deg for 60 minutes
                    1. Purify over zymo Clean and Concentrator 25 column following small RNA removal protocol and elute in 30µL DEPC H2O

                    Quality control: glyoxal gel

                    1. Run a glyoxal gel of all RNAs and verify their size.
                    1. Also glyoxylate the ladder and include (typically I use 5µL of either of the ladders listed above)
                    1. Use Image Lab to quantify the full-length band and use this to determine concentration in combination with nanodrop readings.
                    1. If significant truncated products are present I recommend agarose gel purification of full-length RNA using the Zymo Agarose Gel Zymoclean kit (R1011)
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