We are running a firefly luciferase assay on two cell lines (Huh & HAP-1) transiently expressing various RNAs containing the firefly luciferase gene. Readings have been extremely variable both between repeated experiments (varying between 10000 and >250000 for the same RNA in the same cell line) but also more importantly between repeated readings of the same lysate from a single well. The kit we are using is Promega e1501 but with the renilla lysis buffer.

Any advice would be much appreciated as we have tried varying almost all our protocol. Assays have most often been performed manually but even when using a luminometer which automatically adds the luciferase results have been poor.