Protein synthesis in eukaryotes is subject to stringent control. Misregulation of translation of certain mRNAs is often a hallmark of many diseases, including malignancies and autoimmune disorders. To understand why and how it happens, it is important to investigate the translational control of specific mRNAs. In this case, one could use reporter mRNAs in order to identify cis-acting elements responsible for regulation. Here we overview plasmid DNA (pDNA) and mRNA transfections, their pitfalls and limitations, as well as some emerging applications for mRNA transfection.