Luciferase assay reagent 2 lar 2

The Dual-Luciferase reporter assay system (Promega) was used to measure the luciferase activity of cells cotransfected with wild-type and mutant PDCD4 3′-UTR vectors in combination with miRNA mimics. Firefly and luciferase activities were measured 24 h after transfection. The cells were washed with a phosphate-buffered saline (PBS) solution and lysed with 100 μl of passive lysis buffer (Promega) for 15 min at room temperature. The contents of each well were then freeze-thawed to complete lysis, and 20 μl of the lysate was added to individual wells of an opaque 96-well plate (PerkinElmer). This was followed by the addition of 100 μl of luciferase assay reagent II (LARII) (Promega). The Tecan Infinite 200 Pro spectrophotometer was then used to measure firefly activity. After this measurement, 100 μl of Stop & Glo reagent (Promega) was added to each well, and Renilla activity was measured. For each luminescence reading, there was a 2-s premeasurement delay, followed by a 10-s measurement period. Analyses of all samples were performed in triplicates, and luciferase activity was expressed as a Renilla/firefly ratio to normalize for cell number and transfection efficiency. Each transfection condition was calculated as a ratio of the control let-7a luciferase activity. This calculation was separately applied to each of the different mutant transfection conditions.

Cell lysates transfected with pGL3 vector constructs were prepared as described above, except the lysis solution was 1× Passive Lysis Buffer (PLB, Promega, Madison, WI, USA). Cell lysates were brought to room temperature, of which 20 μL was used to carry out the luciferase assays by the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions, using 50 µL Luciferase Assay Reagent II (LAR II) (Promega, Madison, WI, USA) and 50 μL Stop & Glo Reagent (Promega, Madison, WI, USA). The measurements were carried out as described above, and sequential readings for firefly luciferase and Renilla luciferase were recorded.

The seeding of HEK293T cells took place into 96-well plates and underwent culture to 50–70% confluence prior to transfection. For circRBMS3 and miR-424, 600 ng plasmids of circRBMS3-wt and circRBMS3-mut, 20 nmol miR-424 and N.C. were transfected. Following incubation for 48 hours, the detection of firefly and Renilla luciferase activities was conducted by the Promega Dual-Luciferase system. For providing an internal reference, firefly luciferase activities were evaluated with a 100 ml Luciferase Assay Reagent II (LAR II) (Luciferase Assay Reagent, Promega), and then 20 ml lysis buffer, moreover 100 ml Stop and Glo® Reagent (Luciferase Assay Reagent, Promega) was utilized for evaluating Renilla luciferase activities. Calculation of the difference between a firefly and Renilla luciferase activities was to evaluate relative luciferase activity.