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  • HiScribe™ T7 ARCA mRNA Kit (with tailing)

    Description

    Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´ end and a poly(A) tail at the 3´ end to be efficiently translated. The HiScribe T7 ARCA mRNA Kit (with tailing) is designed for quick production of ARCA capped and poly(A) tailed mRNA in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA, NEB #S1411) using T7 RNA Polymerase. The transcription reaction can be set up easily by combining the ARCA/NTP mix, T7 RNA Polymerase Mix and a suitable DNA template. The kit also allows for partial incorporation of 5mCTP, Pseudo-UTP and other modified nucleotides into mRNA. After a brief DNase I treatment to remove the template DNA, capped mRNA is poly(A) tailed with Poly(A) Polymerase. mRNAs synthesized with the kit can be used for cell transfection, microinjection, in vitro translation and RNA vaccines.

    ARCA is incorporated into mRNA exclusively in the correct orientation, generating capped mRNA that is more efficiently translated. Standard cap analogs can be incorporated in either direction resulting in only 50% of capped mRNA that is functional in protein translation.

    Figure 1. Structure of Anti-Reverse Cap Analog (ARCA, NEB #S1411)

    Methylation at the 3´ position of 7mG forces the cap structure to be attached to mRNA in the correct orientation.
    Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA Kit (with tailing)Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA Kit (with tailing)

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ARCA/NTP Mix-202X
    CLuc Control Template-200.25 µg/µl
    DNase I (RNase-free)-202 units/μl
    Poly(A) Polymerase Reaction Buffer-2010X
    LiCl Solution-20
    E. coli Poly(A) Polymerase-20
    T7 RNA Polymerase Mix-20

    Properties and Usage

    Materials Required but not Supplied

    • DNA template
    • Thermocycler or 37°C incubator.
    • Nuclease-free water
    • Buffer- or water-saturated phenol:chloroform
    • Ethanol
    • 3 M Sodium acetate, pH 5.2
    • 5 M Ammonium acetate
    • Spin columns
    • Gels, running buffers and gel box
    • Equipment for RNA analysis

    Storage Temperature

    -20°C

    Troubleshooting

    Troubleshooting

    • Control Reaction
      The CLuc control template DNA is a linearized plasmid containing the Cypridina luciferase gene under the transcriptional control of the T7 promoter. The size of the runoff transcript is 1.6 kb. The control reaction should yield ≥ 15 μg RNA transcript in 30 minutes.

      If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take all precautions to avoid RNase contamination. Contact NEB for technical assistance.

      The control plasmid sequence can be found here. The CLuc control template is generated by linearizing the plasmid with restriction enzyme Xba I.

    • Low Yield of Full-length RNA
      If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol:chloroform extraction is recommended.

    • Low Yield of Short Transcript
      High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield.

    • RNA Transcript Smearing on Denaturing Gel
      If the RNA appears degraded (e.g., smeared) on denaturing agarose or polyacrylamide gel, the DNA template is likely contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (a smear below the expected transcript length). We recommend evaluating the plasmid DNA template with the RNase Contamination assay Kit (NEB #E3320). If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water.

    • RNA Transcript of Larger Size than Expected
      If the RNA transcript appears larger than expected on a denaturing gel, plasmid DNA may be incompletely digested. Even small amounts of undigested circular plasmid DNA can produce large amounts of long transcripts. Check template for complete digestion. If undigested plasmid is confirmed, repeat restriction enzyme digestion.

      Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structures.

    • RNA Transcript of Smaller Size than Expected
      If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase. Sequences with resemblance to T7 RNA Polymerase termination signals will cause premature termination. Incubating the transcription reaction at lower temperatures, for example at 30°C, may increase the proportion of full-length transcript, however the yield will be decreased. For GC rich templates, or templates with secondary structures, incubation at 42°C may improve yield of full-length transcript.

    • Tailing Length Control
      A standard 30 min tailing reaction can add a poly(A) tail at least 150 nt in length to an average size mRNA generated from the IVT reaction. Short RNA may require longer incubation time for sufficient tailing.

    • No Tailing or Partial Tailing
      3´ end of the mRNA must be exposed for efficient tailing. Because T7 RNA Polymerase tends to generate 3´ end heterogeneity by adding extra bases, a small percentage of the mRNA may adopt alternate structures which may not be suitable for tailing. The following tips may help with successful tailing.
      • Run the whole mRNA synthesis work flow without freezing the RNA between steps.
      • To avoid preferential tailing, pre-incubate tailing mix at 37°C for 3 minutes before adding Poly(A) Polymerase. Mix well immediately.
      • Tailing reaction should be at 37°– 40°C. Lower temperatures are not recommended.
      • If still no tailing, redesign the DNA template with different sequences at the 3´ end.

    • mRNA not functional
      • Verify the mRNA is intact, capped and tailed.
      • Be sure the mRNA is clean, free from any inhibitors of downstream experiments.
      • Follow instructions carefully with appropriate controls.
      • Verify the DNA template has the correct sequence.

    Notes

    1. All kit components should be stored at –20°C. The kit contains sufficient reagents for 20 reactions of 20 μl each. Each standard reaction yields up to 20 μg of capped mRNA from 1 μg control template. Up to 25 μg capped and tailed mRNA can be obtained after Poly(A) tailing and purification by LiCl precipitation.

    FAQs

    1. HiScribe™ T7 ARCA mRNA Kit (with tailing) What is the difference between the HiScribe T7 ARCA mRNA Kit (NEB #E2065) and the HiScribe T7 ARCA mRNA Kit (with Tailing)(NEB #E2060)?
    2. I currently use mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use?
    3. I currently use MessageMAX™ T7 ARCA-capped Message Transcription Kit, which mRNA synthesis kit from NEB should I use?
    4. Can modified nucleotides be used with the HiScribe T7 ARCA mRNA kits?
    5. What is the difference between the HiScribe T7 ARCA mRNA kits and the HiScribe T7 High Yield RNA Synthesis Kit (E2040) and HiScribe T7 Quick RNA Synthesis Kit (E2050)?

    Protocols

    1. Standard mRNA Synthesis (E2060)
    2. mRNA Synthesis with Modified Nucleotides (E2060)
    3. mRNA Purification (E2060)
    4. Evaluation of Reaction Products (E2060)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.