引领高效 RNA 合成：NEB 发布规模化 IVT 技术参考指南

图1：Gentle, intermittent mixing during IVT incubation maximizes yields.

HiScribe reactions (1 ml, 5 kb transcript, expected yield = 9 mg) were incubated for 1 hour at 37°C in a dry air incubator and were either not mixed, mixed after 30 minutes, or mixed every 15 minutes by gentle inversion. Care was taken to ensure no bubbles were introduced during mixing.

图2：T7 RNA Polymerase titration indicates enzyme amount required to reach maximum yields and demonstrates scalability.

A. In vitro transcription reactions (per 20 µl reaction: 1X RNA Pol Buffer, 5 mM each NTP, 20 mM MgCl2 (total), 0.1 units Inorganic Pyrophosphatase (E. coli) at 37°C for 2 hours) were performed with increasing units of T7 RNA Polymerase to determine the amount of enzyme needed to reach maximum yields. A minimum of 100 units of T7 RNA Polymerase is required to transcribe ~100 µg of CLuc RNA. B. Reaction conditions scaled linearly from 20 µl to 10 ml results in linear RNA yields.

图3：IVT reactions can be scaled linearly when using HiScribe kits or individual reactions.

IVT reactions (5 and 9 kb transcripts) were scaled linearly from 20 µl to 20 ml with A. HiScribe components (10 mM each NTP), B. high yield and individual enzymes with 10 mM each NTP, and C. moderate yield and individual enzymes with 5 mm each NTP. R2 > 0.99 for all reactions.

NEB 生产的 HiScribe 高效体外转录试剂盒和多种酶类工具（包括 T7 RNA 聚合酶、无机焦磷酸酶和 RNase 抑制剂）均提供 RUO 和 GMP 级别，为合成各种 RNA 提供了坚实的原材料基础。为了增强 RNA 的稳定性并生产完全功能的 mRNA，NEB 为科学家提供了多种策略的解决方案，包括转录后的 Poly (A) 聚合酶添加、共转录时的加帽或使用 Faustovirus 或 Vaccinia 加帽酶进行转录后加帽，以及加入修饰核苷酸以减少免疫反应。

随着 RNA 作为治疗手段的兴起，NEB 期望本研究指南中的建议能为客户提供 IVT 产量线性放大的思路和方法，帮助他们使用超稳定且一致性佳的试剂，这些试剂可以帮助客户合成大量高质量的 RNA。

NEB 致力于支持 mRNA 合成，提供从模板到转录的全套工具，与您携手共创科技新篇章。