Passive lysis buffer dual luciferase reporter assay system

PsiCHECK-2/HIF3α-short and psiCHECK2/HIF3α-long constructs were transfected using Fugene HD transfection reagent (Promega) 24 h after cell seeding, or when mimics were used, 24 h after miRNA mimic transfection, according to recommendations by the manufacturer. After 24 hours the cells were washed with PBS and lysed by Passive Lysis Buffer (Dual Luciferase® Reporter Assay System, Promega) for 30 min on a shaker platform. Lysates were transferred to white 96-wells microplates. Luciferase Assay Reagent II (Dual Luciferase® Reporter Assay System, Promega) was added, immediately followed by quantitation of the firefly luciferase activity on a luminometer (Aspekt Fluoroskan). Subsequently, Stop & Glo® Reagent (Dual Luciferase® Reporter Assay System, Promega) was added to the mixture, immediately followed by quantitation of the Renilla luciferase activity. Relative luciferase signals of duplicates were averaged, values (n = 3) were normalized, and average and standard deviations were calculated. Significant differences in luciferase activity were determined by two-sample t-tests.

The DNA sequence of the Myomaker enhancer was synthetized by IDT and PCR amplified using GXL polymerase. The enhancer was cloned into a pGL4.Luc plasmid using KpnI and HindIII restriction enzymes. PE cells were transfected with the Enhancer reporter construct, CMV-Lacz plasmid and plasmids encoding the TFs in triplicate. Cells were lysed in Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, Promega) and frozen for 15 min at −80 °C. After thawing, cells were kept on ice and lysates were resuspended 7 times by pipetting. Lysates were transferred into a 96 well plate (white bottom) with of reconstituted renilla luciferase (from Promega). renilla luciferase activity was measured by plate reader (BMG Labtech, CLARIOstar). After measurement, lysates were stained against Beta-galactosidase and quantified with the same plate reader. Beta-galactosidase measurements were normalized for all conditions to the negative control. Then luciferase signal was normalized to the normalized Beta-galactosidase value for that condition.