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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:

  1. 产品编号 产品名称 信息
  2. B8174 mCAP m7G(5')ppp(5')G Cap的类似物,用于构建 Cap 0结构
  3. B8175 ARCA 抗反向帽类似物(ARCA),用以形成Cap 0结构,3´-O-Me-m7G(5')ppp(5')G
  4. B7972 Pseudo-UTP
  5. B7967 5-Methyl-CTP
  6. B8061 5-Methoxy-UTP

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


  1. 产品编号 产品名称 信息
  2. R1001 ARCA EGFP mRNA 用ARCA修饰的增强型绿色荧光蛋白的mRNA,具有稳定高效的表达效率,可作为实验对照。
  3. R1002 ARCA EGFP mRNA (5mCTP, ψUTP) 用ARCA, 5mCTP(5-methyl-CTP), ψUTP (pseudo-UTP)修饰的增强型绿色荧光蛋白mRNA, 抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可作为实验对照。
  4. R1003 mCAP EGFP mRNA 使用mCAP修饰的增强型绿色荧光蛋白的mRNA,具有稳定高效的表达效率,可作为实验对照。
  5. R1004 mCAP EGFP mRNA (5mCTP, ψUTP) 使用mCAP, 5mCTP(5-methyl-CTP), ψUTP (pseudo-UTP)修饰的增强型绿色荧光蛋白mRNA, 抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可作为实验对照。
  6. R1005 Firefly Luciferase mRNA (ARCA, 5mCTP, ψUTP) 具备更好性能ARCA、5mCTP和ΨUTP修饰的萤火虫荧光素酶mRNA,抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可作为实验对照。
  7. R1006 SpCas9 mRNA (ARCA, 5mCTP, ψUTP) 应用于基因编辑技术,和guideRNA协同对DNA进行定点切割。
  8. R1007 ARCA EGFP mRNA (5-moUTP) 具备更好性能的5-moUTP修饰mRNA,抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可作为实验对照。
  9. R1008 ARCA Cy3 EGFP mRNA (5-moUTP) 具备更好性能的5-moUTP修饰mRNA,带有Cy3标记,抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可进行定位以及实验对照。
  10. R1009 ARCA Cy5 EGFP mRNA (5-moUTP) 具备更好性能的5-moUTP修饰mRNA,带有Cy5标记,抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可进行定位以及实验对照。
  11. R1010 EZ Cap™ Cy5 Firefly Luciferase mRNA (5-moUTP) 具有Cap 1结构的萤火虫荧光素酶mRNA,使用5-moUTP和Cy5-utp修饰,提供更高的转录效率并抑制RNA介导的先天免疫激活。
  12. R1011 EZ Cap™ Cy5 EGFP mRNA (5-moUTP) 具有Cap 1结构的EGFP mRNA,使用5-moUTP和Cy5-utp修饰,提供更高的转录效率并抑制RNA介导的先天免疫激活。
  13. R1012 Firefly Luciferase mRNA (ARCA, 5-moUTP) 具备更好性能ARCA和5-moUTP修饰的萤火虫荧光素酶mRNA,抑制RNA介导的先天免疫激活,具有稳定高效的表达效率,可作为实验对照。
  14. R1013 EZ Cap™ Firefly Luciferase mRNA (5-moUTP) 具有Cap 1结构的萤火虫荧光素酶mRNA,使用5-moUTP修饰,提供更高的转录效率并抑制RNA介导的先天免疫激活。
  15. R1014 ARCA Cas9 5-moUTP 具有Cap 0结构的Cas9 mRNA,使用5-moUTP修饰增加mRNA的翻译效率和稳定性,降低免疫原性。
  16. R1015 CleanCap Cas9 5-moUTP 应用于基因编辑技术,和guide RNA协同对DNA进行定点切割。