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)
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 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 (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.
Marc Herb, Alexander Gluschko, Katja Wiegmann, Alina Farid, Anne Wolf, Olaf Utermöhlen, Oleg Krut, Martin Krönke, Michael Schramm (2019) Science Signaling; 12(568), eaar5926..
Marc Herb, Alina Farid, Alexander Gluschko, Martin Krönke, Michael Schramm (2019) Highly efficient transfection of primary macrophages with in vitro transcribed mRNA J Vis Exp; e60143..
Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. Please review and update your order accordingly If you have any questions, please contact Customer Service at freezers@neb.com or 1-800-632-5227 x 8.
Help us celebrate our 50th anniversary! We have hidden 1,000 golden butterflies and are waiting for you to find them. They can be anywhere that you find NEB! Beginning April 15th, be sure to visit our website and tables at tradeshows and events you are attending. Visit our social media channels frequently for tips on where we have hidden the butterflies – and once you find one, either click or scan the code to be eligible for a 50th anniversary prize pack, as well as a grand prize trip to NEB headquarters in Ipswich, MA!.
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Institution Changed
Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.
Sign in to your NEB account
To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.
Powered by Bioz
