U251 cell was seeded into 24-well culture plates, reached to around 50% confluency and then co-transfected for 48 hrs with reporter plasmid (200 ng), pRL-CMV-Renilla (Promega, Madison, WI) plasmid (1 ng) and miRNA using Lipopectamine 2000 (Invitrogen). Luciferase activity was measured using dual-luciferase reporter Assay system (Promega) according to the manufacturer's instructions [22 (link)] and normalized to Renilla luciferase activity. All experiments were performed in triplicates.
Lipopectamine 2000
Manufactured by Thermo Fisher Scientific
Lipofectamine 2000 is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of cell types. It facilitates the formation of lipid-nucleic acid complexes that can be efficiently taken up by cells, enabling the introduction of genetic material for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin rna isolation kit, by Takara Bio (2 mentions)
Qscript cdna synthesis supermix, by Quantabio (2 mentions)
Peitransfecting reagent, by Avantor (2 mentions)
Purelink genomic dna kit, by Thermo Fisher Scientific (2 mentions)
Prl cmv renilla, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
4 protocols using lipopectamine 2000
1
Dual-Luciferase Reporter Assay for miRNA
2
CRISPR-Cas9 Mediated AR Promoter Editing
3
siRNA Transfection and Overexpression
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Cells were transfected with siRNA and/or overexpression vector using Lipopectamine 2000 (Invitrogen) or polyethylenimine (PEI) according to the manufacturer’s instructions. After 48 h transfection, cells were harvested. All siRNA was purchased from Genolution Pharmaceuticals, Inc. (Seoul, Korea). All experiments were independently repeated three times with similar results.
4
CRISPR-Cas9 Mediated AR Promoter Editing
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sgRNAs targeting indicated AR promoter regions (Table S5 ) were designed using
the MIT CRISPR Design software (crispr.mit.edu ). Each sgRNA oligos were synthesized and
cloned into lentiCRISPR v2 vector as a gift from Dr. Feng Zhang (Addgene
pladmid #52961). Lentiviral particles was produced in 293T with PEI
transfecting reagent (VWR). LNCaP cells were then infected with sgRNAs
lentiviral particles combination for 48 hours, then split, and transfected
with either control or siEZH2 using Lipopectamine 2000 (Invitrogen) for 48
hours. Genomic DNA was prepared using the PureLink Genomic DNA kit (Life
Technology). PCR of genomic DNA was performed with indicated primers
flanking the sgRNA target sites on AR promoter region (Table S5 ). PCR products were
purified from agarose gel and sequenced to assess the effects of
CRISPR-Cas9-mediated editing of AR promoter. Total RNA was isolated from
cells with Nucleospin RNA isolation kit (Clonetech) and 250 ug of RNA per
sample was used for cDNA synthesis using qscript cDNA synthesis supermix
(Quantabio). PCR of cDNA were then performed using specific AR promoter
(also exon 1) primers (Primer F2 and R2) and subjected for agarose gel
analysis. Protein extracts were subjected for western blot analysis to
confirm EZH2 knockdown.
the MIT CRISPR Design software (
cloned into lentiCRISPR v2 vector as a gift from Dr. Feng Zhang (Addgene
pladmid #52961). Lentiviral particles was produced in 293T with PEI
transfecting reagent (VWR). LNCaP cells were then infected with sgRNAs
lentiviral particles combination for 48 hours, then split, and transfected
with either control or siEZH2 using Lipopectamine 2000 (Invitrogen) for 48
hours. Genomic DNA was prepared using the PureLink Genomic DNA kit (Life
Technology). PCR of genomic DNA was performed with indicated primers
flanking the sgRNA target sites on AR promoter region (
purified from agarose gel and sequenced to assess the effects of
CRISPR-Cas9-mediated editing of AR promoter. Total RNA was isolated from
cells with Nucleospin RNA isolation kit (Clonetech) and 250 ug of RNA per
sample was used for cDNA synthesis using qscript cDNA synthesis supermix
(Quantabio). PCR of cDNA were then performed using specific AR promoter
(also exon 1) primers (Primer F2 and R2) and subjected for agarose gel
analysis. Protein extracts were subjected for western blot analysis to
confirm EZH2 knockdown.
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