Protocol for monitoring mRNA translation and degradation in human cell-free lysates

Cell-free translation is a powerful tool for studying protein synthesis. Here, we present a protocol for monitoring mRNA translation and degradation using a human cell-free translation system. The protocol includes instructions for preparing translation-competent lysates via dual centrifugation, generating and capping reporter mRNAs compatible with luciferase assays, performing cell-free translation reactions, and assessing mRNA stability by northern blotting. The protocol supports comparative analysis of translation efficiency and mRNA decay for various 5' UTRs and viral RNA elements. For complete details on the use and execution of this protocol, please refer to Gurzeler et al.¹ and Bäumlin et al.².