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. By using
a DNA template encoding a poly(A) tail, the HiScribe T7 ARCA mRNA Kit can
be used to synthesize capped and tailed mRNAs. The cap structure is added
to the mRNA by co-transcriptional incorporation of Anti-Reverse Cap Analog
(ARCA) (NEB #S1411) using T7 RNA Polymerase. Poly(A) tail is incorporated
during the transcription reaction. The kit also includes DNase I and LiCl for
DNA template removal and quick mRNA purification.
Additionally, the kit is capable of partial incorporation of modified UTP and
CTP (up to 50% each) without affecting the mRNA yield significantly. By using
a DNA template encoding a poly(A) tail, capped and tailed modified mRNA can
be synthesized in a single reaction in 30 minutes. 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: T7 RNA Polymerase co-transcriptionally caps RNA with anti-reverse cap analog (ARCA)
Figure 3: Overview of mRNA synthesis workflow with the HiScribe T7 ARCA mRNA Kit
This product is related to the following categories:
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 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 (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). 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 structure.
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
Tail length is defined by the poly(A) coding length on the DNA template.
Poly(A) tails longer than 125 nt have minimal effect on enhancing mRNA function.
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.
Citations & Technical Literature
Citations
Product Citation Tool
Quality, Safety & Legal
Quality Control Assays
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.
Specifications & Change Notifications
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]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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.
