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)
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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.
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 within the DNA Sequences and Maps Tool under the name “pCMV-CLuc 2”. The pCMVCLuc 2 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 (see template DNA preparation section).
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. Alternatively, clean up the DNA template using a spin column
based method, Monarch PCR & DNA Cleanup Kit (5 μg), NEB #T1030.
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. If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section).
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.
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Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
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The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
