For luciferase (Luc) activity analysis, osteosarcoma MDR cell lines KHOSMR and U-2OSMR cells were seeded in 96-well plates and 100 ng of Luc-Wee1-3′-UTR reporter vector (SwitchGear Genomics, Menlo Park, CA) and 40 nM of miR-15 precursor or 40 nM of non-specific miR precursor control vectors were co-transfected with Lipofectamine 3000 (Life Technologies). Luciferase activity was measured 48 h after transfection by the LightSwitch Luciferase Assay Reagent™ (SwitchGear Genomics).
Lightswitch luciferase assay reagent
Manufactured by SwitchGear Genomics
Sourced in United States
The LightSwitch Luciferase Assay Reagent is a laboratory equipment used to measure luciferase activity. It provides a luminescent signal that can be used to quantify gene expression levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Luc wee1 3 utr reporter vector, by SwitchGear Genomics (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lightswitch 3 utr reporter goclone constructs of, by SwitchGear Genomics (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna mimic, by Thermo Fisher Scientific (1 mentions)
Glomax microplate luminometer, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Flexstation 3 microplate reader, by Molecular Devices (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Polarstar multidetection microplate reader, by BMG Labtech (1 mentions)
Pgl2 basic vector, by Promega (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Dharmafect 2 reagent, by Horizon Discovery (1 mentions)
Lightswitch luciferase assay system, by SwitchGear Genomics (1 mentions)
Pgfp c shlenti vector, by OriGene (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Fluostar omega plate reader, by BMG Labtech (1 mentions)
25 protocols using lightswitch luciferase assay reagent
1
Luciferase Activity Analysis in Osteosarcoma MDR Cells
2
Assessing miRNA Regulation of 3′-UTR Reporter
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The LightSwitch 3′-UTR reporter GoClone constructs of various putative miRNA targets were purchased from SwitchGear Genomics (Active Motif). The entire 3′-UTRs were placed downstream of a RenSP luciferase reporter under the control of the cytomegalovirus (CMV) promoter, thus the impact of a miRNA on regulation of transcript stability or translation efficiency could be assessed. For the assay, Huh7.5.1 cells were transfected with miRNA mimic of interest or with mimic control for 24 h in 96-well white plates (in five replicates), and then transfected with 50 ng of GoClone 3′-UTR reporter plasmid using FuGENE 6 transfection reagent (Roche). Two days later, cells were lysed and total luciferase outputs were read using LightSwitch Luciferase Assay Reagent (SwitchGear Genomics).
3
Transfection and Luciferase Assay Protocol
4
Investigating miRNA-204-5p Regulation of PPARGC1A
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C2C12 cells were cultured in DMEM (4.5 g/L d ‐glucose) supplemented with 10% fetal bovine serum, 2% HEPES, and 1% minimum essential medium non‐essential amino acids solution and seeded in 24‐well plates at a density of 50,000 cells/well. The next day, cells were transfected with hsa‐miRNA‐204‐5p or Neg control #1 (Ambion; Life Technologies, Waltham) precursor molecules and cotransfected with the LightSwitch_PPARGC1A_3′UTR vector (Active Motif, Carlsbad) using Lipofectamine 2000 (Invitrogen, Waltham). After 24 hr, the luciferase activity was determined using the LightSwitch Luciferase Assay Reagent (SwitchGear Genomics, Carlsbad) according to the manufacturer's instructions using a GLOMAX Microplate Luminometer (Promega Corporation, Madison, WI).
5
Evaluating SNAI2 Promoter Activity
6
NLRP3 Gene Promoter and 3'UTR Analysis
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LightSwitch Promoter and 3’UTR Reporter GoClone plasmid DNA constructs for the NLRP3 gene were purchased from Switchgear Genomics (Product ID S718266 and S805599). HEK cells were transfected with these plasmids and an empty plasmid as per manufacturer protocol using FuGene HD Transfection Reagent (Promega). Lightswitch Luciferase Assay Reagent (Switchgear Genomics) was used prior to luminometry to measure luciferase reporter signals also per manufacturer protocol.
7
Transient Transfection Assay for AURKB
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HEK293 cells were transiently co‐transfected with vectors containing empty promoter or AURKB promoter fused with luciferase reporter (SwitchGear Genomics, Menlo Park, CA) and pLJM1 alone or with pLJM1 vector expressing WT1 using lipofectamine‐3000 transfection reagent (Invitrogen). After 72 h, cells were lysed, and Renilla luciferase activity was measured using LightSwitch Luciferase Assay Reagent (SwitchGear Genomics) in a Flex Station 3 microplate reader (Molecular Devices). Luciferase activity is reported as relative luminescence units (RLU).
8
Validating miR-135a Target Genes
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The LightSwitch 3′ UTR reporter GoClone constructs of various putative miR-135a targets were purchased from SwitchGear Genomics (Active Motif). Huh7.5.1 cells were transfected with miR-135a WT or MUT or with mimic control in 96-well white plates (in 5 replicates). After 24 h, cells were further transfected with 50 ng of GoClone 3′ UTR reporter plasmid using FuGENE 6 transfection reagent (Roche). Two days later, cells were lysed and total luciferase outputs were quantified using LightSwitch Luciferase Assay Reagent, according to the manufacturer’s instructions (SwitchGear Genomics) using a POLARstar multidetection microplate reader (BMG Labtech).
9
STAT3 Promoter Activity Assay
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HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector. Cells were lysed after 48 h and analyzed using LightSwitch Luciferase Assay Reagent (Switchgear Genomics).
10
Regulation of NRP2 Gene Expression
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The 2.3 kb NRP2 gene promoter (with 5′UTR) (−1500 to +791) or 1.5 kb promoter (without 5′UTR) (−1500 to transcriptional start site) were amplified from human AoSMC genomic DNA and then subcloned into an EMPTY_PROM (switchgear genomics, S790005) promoter reporter vector. The 5′UTR (transcriptional start site to +791) of NRP2 was amplified and subcloned into an EMPTY_5′UTR (switchgear genomics, S690005) 5′UTR Reporter Vector. The primer sequences used for cloning insertion are listed in Table S3 . All clones were verified by DNA sequencing. Luciferase assay was performed using the Lightswitch luciferase assay reagent (Switchgear Genomics, LS010) following the manufacturer's instructions. In brief, 24 hours after transfection with luciferase assay plasmid, 5000 AoSMC cells per well were seeded in white 96‐well plates and cultured for 12 hours (no Lipofectamine). Cells were then transfected with an empty vector or Smad3‐overexpressing plasmid (Smad3‐OE) for 24 hours. The cell culture was changed to fresh basal medium and incubated for 12 hours, treated with TGFβ1 (10 ng/mL) for 2 hours, and then used for luciferase assay by adding 100 μL Assay Solution Promega. The plate was incubated at room temperature for 30 minutes before reading in Luminometer System (Applied Biosystems). Luciferase activity was normalized to cell number from duplicate plates.
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