This protocol describes the stepwise synthesis and preparation of capped, polyadenylated messenger RNA (mRNA) suitable for zebrafish microinjection, using T7 in vitro transcription followed by separate enzymatic capping and poly(A) tailing. The workflow begins with the preparation of a DNA template containing a T7 promoter, which is transcribed in vitro using the NEB HiScribe T7 High Yield RNA Synthesis Kit to generate high-yield uncapped RNA. The transcript is subsequently capped using the Faustovirus capping enzyme (FCE) to produce a type 1 5′ cap structure (m⁷GpppNm), which enhances translation efficiency and stability in eukaryotic systems. Following capping, a poly(A) tail is enzymatically added using E. coli poly(A) polymerase to improve RNA stability, nuclear export, and translational competence in zebrafish embryos. Each enzymatic step is followed by RNA purification using lithium chloride precipitation to remove unincorporated nucleotides, enzymes, and buffer components. The protocol includes quality control measures such as spectrophotometric or fluorescence-based RNA quantification, optional gel electrophoresis for integrity assessment, and guidelines for RNase-free handling. Final mRNA products are resuspended in low-salt Tris-HCl buffer (pH 7.5–8.0) and aliquoted for storage at −80 °C to maintain long-term stability. This method provides a robust and flexible approach for generating high-quality synthetic mRNAs with customizable features, suitable for functional studies and developmental biology applications in zebrafish.
James Jam Jolly (Sun,) studied this question.