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RNA

Custom mRNA Synthesis

The ability to synthesize RNA in the laboratory is critical to many techniques. Synthesis of RNA transcripts containing modified nucleotides can be used for various biochemical and molecular biology studies. Large scale transcription reactions, generating up to 200 µg of RNA per reaction can be used for RNA amplification, expression studies (microinjection, infection with viral transcripts, in vitro translation), structural analysis (protein-RNA binding), and mechanistic studies (ribozyme analyses). We can provide milligram scale RNA synthesis service.

Modified Nucleotide-containing mRNA Synthesis

 

 

In Vitro Synthesis of mRNA (In vitro transcription, IVT)

 

A 7-methyl guanosine (m7G) cap structure at the 5´ end and a poly(A) tail at the 3´ end are required for mRNA to be translated efficiently in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) via T7 RNA Polymerase. DNase I is used to remove the template DNA, so Poly(A) Polymerase can attach poly(A) tail to capped mRNA. 5-Methyl-CTP, Pseudo-UTP and other modified nucleotides can also be incorporated into mRNA. Synthetic mRNAs are applicable in cell transfection, microinjection, in vitro translation and RNA vaccines etc.

 

Our custom synthesis mRNA covers a wide range of applications:

  • mRNA for genome editing, e.g. Zinc-finger Nuclease mRNA, TALEN mRNA, Cas9 mRNA and Recombinase mRNA.
  • Reporter gene mRNA, such as EGFP mRNA and Luc mRNA, for fluorescence microscopy, flow cytometry and bioluminescent imaging.
  • Reprogramming mRNA, i.e mRNA for non-integrating generation of iPSC.

 

 

 

Validation:

 

 

Related Products
Cat.No.Product NameCat.No.Product Name
B8175ARCAB8174mCAP
B7972Pseudo-UTP(ψUTP)B79675-Methyl-CTP(5mCTP)

 

 

mRNA Purification

 

mRNAs transcribed in vitro by T7 RNA polymerase may contain various contaminants, such as short RNAs produced by abortive initiation events, double-stranded (ds)RNAs generated by self-complementary 3’extension, as well as unincorporated nucleoside triphosphates, small abortive transcripts and plasmid template. Certain RNA sequences even induce high levels immunogenicity.

  

APExBIO offers purification service to remove the contaminants of modified nucleotide-containing mRNA, thus increase the processing efficiency for downstream applications.

 

Silica-gel Membrane Spin Column Purification:

 

It is a solid phase extraction technique for fast nucleic acid purification. mRNA can be bound to solid phase of silica-gel membranes under certain conditions, with subsequent washing and elution steps in water or TE pH 7. This method eliminates most proteins, DNA and NTPs.

 

HPLC purification by ÄKTA avant system:

 

mRNA can be purified by HPLC (ÄKTA avant system) using column matrix of alkylated non-porous polystyrene-divinylbenzene copolymer microspheres and optimized buffer system, followed by mRNA analyses and mRNA isolation from column fractions.

  

HPLC purification removes dsRNA and other contaminants from in vitro synthesized modified nucleotide-containing mRNAs, yielding mRNA with the high level of translation without generation of immunogenicity or RNA sensor activation.

 

mRNA and long RNA products

 

APExBIO supplies the best quality mRNA and long RNA. This new product lines involve custom synthesis of mRNA and long RNA (up to multiple kilobases) with a wide array of modification services at scales ranging from micrograms to milligrams. The mRNA can be generated from DNA templates provided by our customers or we can provide a full service from the ground up. We offer mCAP or ARCA capping or modified nucleotides implication for all our standard mRNA transcripts.

 

 

All of our mRNA products offer:

 

  • Incorporates an anti-reverse cap analog (ARCA) into the transcript to increase translation efficiency
  • Reduces host cell immune response and enhances stability by incorporating modified nucleotides (5mCTP and ψUTP) and a poly(A) tail
  • Degrades the DNA template after RNA synthesis with DNase
  • Removes the 5’ triphosphates at the end of the RNA with phosphatase to further reduce innate immune responses in mammalian cells
  • Employs a robust clean-up spin column system that delivers high yields of mRNAs that are ready for most downstream applications

 

 

Related Products
Cat.No.Product NameCat.No.Product Name
R1001ARCA EGFP mRNAR1003mCAP EGFP mRNA
R1002ARCA EGFP mRNA (5mCTP, ψUTP)R1004mCAP EGFP mRNA (5mCTP, ψUTP)