Cells were transiently transfected with expression constructs or siRNAs and a luciferase reporter construct with pCMV-LacZ or pRSV-LacZ (Clontech) by using LipofectamineTM 2000 reagent (11668-019; Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instruction with minor modifications. The transfected cells were starved in media containing 5% cFBS for 24 h and then stimulated with androgen. They were lysed in luciferase lysis buffer (0.2 M Tris-Cl [pH 8.0], 0.2% Triton X100, and 1% NP-40) at 25 °C for 15 min. Luciferase activity was then analyzed in Beetle Luciferin (E1603; Promega Co., Madison, WI, USA) by using a Centro XS3 LB960 luminometer (Berthold Technologies GmbH & Co. KG, 75,323 Bad Wildbad, Germany) and normalized to β-galactosidase activity read by a Versa Max microplate reader (Molecular Devices, LLC., San Jose, CA, USA). The duplet siRNA sequences for the silencing study are listed in Table S1 .
Pcmv lacz
Manufactured by Takara Bio
Sourced in United States
PCMV-LacZ is a plasmid vector containing the LacZ gene under the control of the cytomegalovirus (CMV) promoter. LacZ encodes the enzyme β-galactosidase, which can be used as a reporter gene for monitoring gene expression in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Centro xs3 lb 960 luminometer, by Berthold Technologies (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Beetle luciferin, by Promega (1 mentions)
Lb940 plate reader, by Berthold Technologies (1 mentions)
Prl tk renilla, by Promega (1 mentions)
Pcdna3.1 v5 hisa vector, by Thermo Fisher Scientific (1 mentions)
4 protocols using pcmv lacz
1
Androgen-Responsive Luciferase Reporter Assay
2
Luciferase Reporter Assay Normalization
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Each firefly luciferase reporter construct was co-transfected with pCMV-lacZ (Clontech, Palo Alto, CA, USA) encoding β-galactosidase, which serves as an internal control to normalize the transfection efficiency. At 36 h after transfection, cells were lysed to measure the luciferase and β-galactosidase activities, as previously described [27 (link)]. Luciferase activity was normalized to β-galactosidase activity.
3
Characterization of miRNA's Effect on Renilla Luciferase
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The reporter plasmids phRL-TK rs2734647C, or rs2734647T, respectively, were constructed by cloning a 3′UTR fragment of 346 bp and including the SNP downstream of the Renilla luciferase open reading frame, making use of the XbaI restriction site of phRL-TK (forward primer: 5′-ATTATCTAGACCAGGTCTACCCCTCCCGGC-3′, reverse primer: 5′-ATTATCTAGAGGCTGCTCCCTGTCCCAGGT-3′). Sequence integrity was verified using Sanger sequencing. Of the Renilla luciferase reporter construct (phRL-TK rs2734647C, or rs2734647T, respectively), 1 μg (per well), plus 1 μg (per well) of the reference construct pCMV-LacZ (Clontech, Mountain View, CA, USA) were co-transfected in the presence of 10 pg of mirVana miRNA mimic hsa-miR-4711-3p, hsa-miR-511, hsa-miR-515-3p, hsa-miR-519e-3p, negative control #2 (all Life Technologies, Darmstadt, Germany), or no miRNA, respectively. After 24 h, cells were split in 96-well plates, creating four technical replicates for each condition, and separately for luciferase and beta-galactosidase measurement. Enzyme activity was determined using a Mithras LB 940 Plate Reader (Berthold, Bad Wildbad, Germany). Renilla luciferase activity was normalized to beta-galactosidase activity.
4
High-Throughput Nuclear Receptor Screening
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Promoters were tested for regulation by individual ligand-activated NR-members that heterodimerize with RXR (Table S1 ). For high-throughput screening, plasmids of NR/RXR heterodimers (15 ng NR + 15 ng RXR), promoter-reporters (30 ng) and internal controls (5 ng) pRL TK-Renilla (Promega, WI, USA) or pCMV-lacZ (Clontech CA, USA) were aliquoted in a volume of 10 μl/well into 384-well plates (Corning, 3707) using a 96-head liquid handling robot (Packard Evolution). The empty pcDNA3.1/V5-HisA vector (Invitrogen) was used to equalize the total amount of DNA transfected in all conditions. For reverse transfection, FuGENE HD (0.195 µl/well) and OptiMEM (4.805 µl/well) were mixed and 5 µl was added per well. After adding, the plate was gently shaken at RT and 35 µl of CV-1 cells (resuspended in phenol red-free DMEM medium with 10% charcoal-stripped serum) was added per well using a Titertek Multidrop 384 automated HT cell dispenser, making up a total volume of 50 µl per well. Plates were covered with a breathable seal and shaken gently before returning them to the incubator. The next day, 5 µl of ligand (Table S1 ) or control medium was added per well and incubated for another 24 h. The third day, plates were analyzed for luciferase activity using a luminometer (Wallac 1420 VICTOR2™ V, Perkin Elmer).
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