A Luciferase-Based Assay for Assessing Cap-Independent Translation in Wheat Germ Extract

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here, we describe a cell-free protocol to quantitatively assess cap-independent translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis, and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of translation activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key features • Stringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. • Evaluation of cap-independent translation suitable for testing combinations of IRES and CDS. • Translation analysis without radioactive labeling.