Lightswitch assay

Manufactured by SwitchGear Genomics

The LightSwitch Assay is a laboratory equipment product designed to facilitate the detection and quantification of gene expression. It provides a reliable platform for analyzing transcriptional activity in various biological samples.

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Lab products found in correlation

Synergy 2 multi detection microplate reader, by Agilent Technologies (1 mentions) Pegfp n3 plasmid, by Takara Bio (1 mentions)

Spelling variants of this product

LightSwitch Luciferase Assay Reagent (25 citations) LightSwitch Luciferase Assay System (23 citations) LightSwitch Luciferase Assay Kit (22 citations) LightSwitch Assay Reagent (16 citations) LightSwitch Assay System (5 citations) LightSwitch Assay kit (5 citations) LightSwitch Dual Assay System (3 citations) LightSwitch Luciferase® Assay System kit (2 citations) LightSwitch Dual Assay System kit (2 citations) Lightswitch Dual Assay kit (2 citations)

4 protocols using lightswitch assay

Evaluating miRNA Regulation of 3'UTR Reporters

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HCT116 parental cells were plated in 24-well plates at 0.12 × 10⁶ cells per well and transfected with 300 ng/μl per well of GoClone 3′UTR reporter constructs (PAM 3′ UTR, FOS 3′ UTR and EHF 3′UTR (SwitchGear Genomics, Menlo Park, CA) and 50 nM of miRNA mimics (miR-181-d and miR-182-3p) or non-targeting miRNA controls using DharmaFect Duo transfection reagent, according to the manufacturers' instructions. The following controls were used: control plasmid encoding the 3′UTR sequence of the housekeeping gene glyceraldehyde 3′-phosphate dehydrogenase (GAPDH) and the Random 3′UTR plasmid, containing non-conserved, non-genic and non-repetitive human genomic fragments. Twenty-four hours after transfection, plates were removed from incubator, rinsed once with PBS and analyzed using the manufacturers' recommended protocol (LightSwitch Assay, SwitchGear Genomics). Luminescence signal was detected using a luminometer (Synergy 2 Multi-Detection Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT). Each experiment included transfections of miRNA mimics and control plasmid in triplicates.
Normalization was done according to the manufacturers' recommendations below: http://switchgeargenomics.com/sites/default/files/pdf/LightSwitch_3UTRnorm.pdf. Statistical analysis was done in each experiment independently.

Sells E., Pandey R., Chen H., Skovan B.A., Cui H, & Ignatenko N.A. (2017). Specific microRNA–mRNA Regulatory Network of Colon Cancer Invasion Mediated by Tissue Kallikrein–Related Peptidase 6. Neoplasia (New York, N.Y.), 19(5), 396-411.

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GPNMB Promoter Activation Assay

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Human GPNMB promoter construct in a Renilla reniformis luciferase reporter gene (Ren SP) vector (LightSwitch Assay, SwitchGear Genomics, Menlo Park, CA) was used to determine differences in GPNMB promoter activation following ARSB silencing by siRNA and exposure to the SHP2 inhibitor PHSP1 (30μM, Sigma-Aldrich), either alone or in combination. The β-actin promoter (GoClone^TM) construct with Renilla luciferase reporter was used as a positive control, and a scrambled sequence (R01) with Renilla luciferase reporter was the negative control. These controls showed the effectiveness of transcription and the specificity of the reactions. Transfections were performed when cells were 60–70% confluent, following silencing or treatment for 24 h, using FuGENE HD transfection reagent and the proprietary LightSwitch Assay reagent (SwitchGear). After incubation for 24 h, luminescence was measured at 480 nm in a microplate reader (FLUOstar, BMG Labtech) and compared among the different cell preparations.

Bhattacharyya S., Feferman L, & Tobacman J.K. (2016). Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF. PLoS ONE, 11(4), e0153463.

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CSPG4 Promoter Regulation in Melanocytes

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The CSPG4 promoter sequence with an Sp1 binding site (GCCCCGCCCC) was identified using online resources [alggen-promo. http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3]. The promoter construct was tagged with a Renilla renformis luciferase reporter gene (RenSP) to detect activation of the promoter following silencing of ARSB, or other treatment in the melanocytes (LightSwitch Assay, SwitchGear Genomics, Menlo Park, CA). The β-actin promoter (GoClone^TM) construct with RenSP was the positive control, and a scrambled sequence (R01) with RenSP was the negative control; these were used to determine the effectiveness and specificity of the transfections. Transfections were performed with cells at 70% confluence, following silencing for 24 h, with FuGENE HD transfection reagent and proprietary LightSwitch Assay Reagent (SwitchGear). Luminescence was read at 480 nm in a microplate reader (BMG) after incubation for 24 hours, and compared among the different cell preparations.

Bhattacharyya S., Feferman L., Terai K., Dudek A.Z, & Tobacman J.K. (2016). Decline in arylsulfatase B leads to increased invasiveness of melanoma cells. Oncotarget, 8(3), 4169-4180.

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Androgen Receptor Luciferase Assay

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AR promoter luciferase reporter assay was performed according to the manufacturer’s protocol (LightSwitch Assay from Switch Gear Genomics, CAT^#LS010). The reporter construct relies on RenSP luciferase driven by the AR promoter. Wild type CDK11 in pCMV6 vector (RC216465) was purchased from Origene (Rockville, MD). pEGFP-N3 plasmid was purchased from Clontech (Mountain View, CA) and served as control. KHOS and U-2OS cell lines (3*10⁴ cells per well) were seeded in 96-well plates. One day post seeding, cells were cotransfected with CDK11 plasmid (20 ng or 40 ng) and GoClone AR reporter (50 ng, Switch Gear Genomics, Prod ID S714892). To test the effect of CDK11 knockdown, KHOS and U-2OS cells were transfected with CDK11 siRNA (10 nM or 20 nM) during cell seeding. Twenty-four hours later, GoClone reporter carrying wild type AR promoter (50 ng,) was transfected in those cells. Luciferase activity with LightSwitch Assay Reagents was measured with a plate luminometer 48 hours after reporter transfection.

Liao Y., Sassi S., Halvorsen S., Feng Y., Shen J., Gao Y., Cote G., Choy E., Harmon D., Mankin H., Hornicek F, & Duan Z. (2017). Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11. Scientific Reports, 7, 43941.

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