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.