title: Affordable luciferase reporter assay for cell-based high-throughput screening.
abstract: The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z' factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: Siebring-van Olst
authors.forename: Christie
authors.initials: C
authors.lastname: Vermeulen
authors.forename: Renee X
authors.initials: RX
authors.lastname: de Menezes
authors.forename: Michael
authors.initials: M
authors.lastname: Howell
authors.forename: Egbert F
authors.initials: EF
authors.lastname: Smit
authors.forename: Victor W
authors.initials: VW
authors.lastname: van Beusechem
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D003065:Coenzyme A / Q000378:metabolism; D003470:Culture Media; D017930:Genes, Reporter; D015894:Genome, Human / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D049409:Luciferases, Firefly / Q000378:metabolism*; D049449:Luminescence; D034622:RNA Interference; D015203:Reproducibility of Results
chemical_list: D003470:Culture Media; D007202:Indicators and Reagents; D049409:Luciferases, Firefly; D003065:Coenzyme A