I’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!
Luciferase assays are useful tools for studying a wide range of biological questions. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. However, once this reagent has been added, how long you wait to measure the signal becomes a key consideration in generating consistent data. Dependent on which luciferase assay you use, you may need a luminometer that can use injectors to deliver the assay reagents. The reason for this is simple, but can be confusing to new users.
A IRES (internal ribosome entry site) is a nucleotide sequence that allows for the translation initiation in the middle of a message RNA sequence as part of the greater process of protein synthesis. Typically an IRES is located in the 5’ UTR of RNA viruses and enables translation of the RNAs in a cap-independent manner. (1,2)
Evaluating a particular RNA sequence for IRES activity relies on designing a bicistronic report construct. When an IRES segment is located between two reporter gene (e.g., Chloramphenicol acetyltransferase [CAT] and Luciferase) open reading frames, it can drive translation of the downstream protein coding region independently from the 5´ cap structure bound to the 5´ end of the mRNA (3,4,5). Continue reading “Characterizing IRES Elements using Cell-Based and Cell-Free Expression Systems”