Glomax 96 well microplate luminometer

Coupled in vitro transcription-translation reactions were carried out in rabbit reticulocyte lysate supplemented with [³⁵S]methionine using a Promega TNT system according to the manufacturer’s instructions. SDS-PAGE followed by autoradiography was performed according to standard procedures. Immunoprecipitations were performed as previously described (71 (link)). Transfection-based reporter assays to assess host cell shutoff by PA-X (described previously [30 (link)]) were performed by cotransfecting QT-35 cells with a reporter plasmid containing the Renilla luciferase gene along with pHW2000 plasmids expressing the appropriate segment 3 genes with or without the desired PA-X mutations. At 48 h posttransfection, cells were lysed, and luciferase activity was measured on a Promega GloMax 96-well microplate luminometer using the Promega Renilla luciferase system.

SH-SY5Y cells were seeded at approximately 7 × 10⁴ cells per well in 24-well plates. After overnight incubation, cells were 70% confluent and were co-transfected with 1 μg reporter plasmid DNA and 20 ng pMLuc-2 (a thymidine kinase promoter driving renilla luciferase vector used as an internal control; Novagen) using TurboFect Transfection Reagent (ThermoScientific/Fermentas), according to manufacturer's protocol.
Luciferase reporter assays were performed 48-hour post transfection. Luciferase activity of reporter constructs was measured using a Dual Luciferase Reporter Assay System (Promega) using 20 μl lysate from transfected cells according to manufacturer's instructions. Assays were carried out on a Glomax 96-well microplate luminometer (Promega). Measurements were averaged from 4 replicates (3 biological replicates assayed in 4 technical replicates). Statistical analyses were performed using two tailed t-tests to calculate significance of the fold change in luciferase expression, compared to the pGL3b backbone vector. To address efficacy of transfection pGL3p and pGL3c luciferase constructs were also included as controls.

The 3’ UTR sequence of NFKBIB was cloned downstream of the firefly luciferase gene. The transcript level is regulated by its interaction with miRNAs resulting in reduced luciferase activity that is measured by the Promega Dual Luciferase Assay (Madison, WI, USA). NFKBIB 3’ UTR reporter plasmid was synthesized by Origene (Origene). HBEpCs cells were transfected on a 96-well plate with a complex containing transfection agent (Lipofectamine 2000, Life Technologies), NFKBIB UTR reporter plasmid, and 25 nM of either the miR-4776 mimic or negative control (a scrambled oligonucleotide that is not a target of any gene). After 18 h of transfection, the medium was replaced with complete medium and the cells were incubated for an additional 16–18 h. Cells were then collected and lysed in lysis buffer supplied by the manufacturer. Luciferase activity was assessed in microtiter plates using the Promega Dual Luciferase Reporter Assay system according to the manufacturer’s instructions (Dual-Glo ® Luciferase Assay, Promega). The plates were read on a Glomax ® 96-well microplate Luminometer (Promega). The relative Renilla luciferase (Promega) activity was calculated by normalizing transfection efficiency to the firefly luciferase activity.

SK-N-AS cells were co-transfected with test constructs (firefly luciferase reporter gene) and an internal control construct, pMLuc-2 (renilla luciferase reporter gene; Novagen, USA) using TurboFect Transfection Reagent (ThermoScientific/ Fermentas, R0531) according to manufacturer's protocol in 24-well plate format. Transfectant was removed after 4 hours of incubation and exchanged with fresh medium and subsequent luciferase activity assays performed after 48 hours of incubation.
Luciferase activity of reporter constructs was measured using a Dual Luciferase Reporter Assay System (Promega, USA) using lysates from transfected cultured cells according to manufacturer's instructions. Assays were carried out on a Glomax 96-well microplate Luminometer (Promega, USA) using 20 µl of cell lysate. Measurements were averaged from 6-fold replicates to minimize pipetting errors and repeated at least three times to confirm results. Statistical analyses were performed using MSExcel software and a one tailed t-test to measure the significance of fold activity of the FUS SVA and TR/VNTR over the minimal promoter of the pGL3P vector *P<0.05, ***P<0.001, and to compare the activity of the alleles of the SVA and the TR/VNTR to each other # P<0.05.

SH-SY5Y cells were seeded at approximately 100,000 cells per well in 24-well plates. After overnight incubation, cells were co-transfected with 2 μg reporter DNA and 20 ng pMLuc-2 (a TK renilla luciferase vector used as an internal control for normalisation; Novagen, USA) using TurboFect Transfection Reagent (ThermoScientific/Fermentas), according to manufacturer’s protocol.
Luciferase reporter assays were performed 48 h post-transfection. Luciferase activity of reporter constructs was measured using a Dual Luciferase Reporter Assay System (Promega) using 20 μl lysate from transfected cells according to manufacturer’s instructions. Assays were carried out on a Glomax 96-well microplate Luminometer (Promega). Two-tailed t test for significance compared fold change in luciferase expression to the vector containing the minimal promoter alone (*P < 0.1, **P < 0.01, ***P < 0.001) N = 4.