RNA_Product_Hero

RNA Synthesis Kits

Return to RNA Synthesis

In vitro RNA synthesis requires a DNA template, RNA polymerase, NTPs and other factors. High-yield robust reactions require optimization of each reaction component. NEB offers five in vitro RNA synthesis kits, all of which have been optimized to generate reproducible yields of quality RNA.

For efficient translation to occur, most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5′ end and a poly(A) tail at the 3′ end. The HiScribe™ T7 ARCA mRNA Synthesis Kit (NEB #E2060S) is designed to synthesize capped and tailed mRNAs for variety of applications. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog, ARCA, using T7 RNA Polymerase generating a Cap-0 structure. A poly(A) tail is then added by E. coli Poly(A) Polymerase. This kit is also available without E. coli Poly(A) Polymerase (NEB #E2065S) for use with DNA templates encoding a poly(A) stretch or not requiring a poly(A) tail. Both kits include DNase I and LiCl for DNA template removal and quick mRNA purification.

The Cap-1 structure has been reported to enhance mRNA translation efficiency (8) and hence may help improve expression in mRNA transfection and in microinjection experiments.  Cap-0 transcripts can be enzymatically converted to cap-1 in vitro. mRNA Cap 2 ́-O-Methyltransferase (NEB #M0366) adds a methyl group at the 2 ́-O position of the first nucleotide adjacent to the cap structure at the 5′ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap-0) resulting in a Cap-1 structure. Alternatively, Cap-1 mRNA can be synthesized co-transcriptionally with a trinucleotide cap analog such as CleanCapâ Reagent AG. The use of CleanCap reagent AG results in significant advantages over traditional dinucleotide co-transcriptional capping. CleanCap Reagent AG is a trinucleotide with a 5′-m7G joined by a 5′-5′ triphosphate linkage to an AG sequence. The adenine has a methyl group on the 2′-O position. The incorporation of this trinucleotide in the beginning of a transcript results in a Cap-1 structure.  The Hiscribe™ T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080S) is formatted with individual vials of NTPs and CleanCap Reagent AG to allow for partial or complete substitution of modified NTPs, with a total kit yield of 1.8 mg of mRNA.  Cap-1 mRNA synthesized from this kit is suitable for many applications, including transfections, microinjections, in vitro translation, preclinical mRNA therapeutic mRNA studies as well as RNA structure and function analysis.

Reference: Kuge, H., et al. (1998) Nucleic Acids Res, 26, 3208–3214.

CLEANCAP® is a registered trademark of TriLink BioTechnologies, LLC.


 
T7 KITS
  SP6 KITS

APPLICATION HISCRIBE
T7 HIGH
YIELD RNA
SYNTHESIS
KIT (#E2040)
HISCRIBE
T7 QUICK
HIGH YIELD
RNA
SYNTHESIS
KIT
(#E2050)
HISCRIBE
T7 ARCA
MRNA KIT
(#E2065)
HISCRIBE
T7 ARCA
MRNA KIT
(WITH TAILING)
(#E2060)
HISCRIBE
T7 MRNA KIT WITH CLEANCAP® 
REAGENT AG  (#E2080)
HISCRIBE
SP6 RNA
SYNTHESIS KIT
(#E2070)
PROBE LABELING

Fluorescent labeling: FAM, Cyanine (Cy) dyes, etc.
• Fluorescent in situ hybridization (FISH)

       

Non-fluorescent labeling: Biotin, Digoxigenin
In situ hybridization
• Blot hybridization with secondary detection
• Microarray

       
High specific activity radiolabeling
• Blot hybridization
• RNase protection
       
  Streamlined high yield CleanCap Reagent AG capped RNA synthesis
• Template encoded poly(A) tails
• Non polyadenylated transcripts
• Transfection
• Microinjection
In vitro translation
           
mRNA & RNA FOR
TRANSFECTION
Streamlined mRNA synthesis with ARCA co-transcriptional
capping and enzymatic poly(A) tailing
• Transfection
• Microinjection
In vitro translation
       
Streamlined ARCA capped RNA synthesis
• Template encoded poly(A) tails
• Non polyadenylated transcripts
• Transfection
• Microinjection
In vitro translation
       
Co-transcriptional capping with alternate cap analogs
• Transfection
• Microinjection
In vitro translation
       
Post-transcriptional capping with Vaccinia mRNA Capping System
• Transfection
• Microinjection
In vitro translation
     
Complete substitution of NTPs: 5-mC, pseudouridine, etc.
• Post-transcriptional capping with Vaccinia mRNA Capping System
       
Partial substitution of NTPs: 5-mC, pseudouridine, etc.    
Unmodified RNA        
Hairpins, short RNA, dsRNA
• Gene knockdown
       
STRUCTURE,
FUNCTION,
& BINDING STUDIES
Complete substitution of NTPs
• Aptamer selection
• Isotopic labeling
       
Partial substitution of one or more NTPs
• Aptamer selection
• Structure determination
         
Unmodified RNA
• SELEX
• Structure determination
       


Learn about RNA Synthesis