hTCF and hTMC were transiently transfected with 150 pmol per 1 × 106 cells specific siRNA (ON-TARGETplus SMARTpool; GE Healthcare Dharmacon; Freiburg, Germany) against RARA or RXRA by electroporation using the Nucleofector II transfection device and the Amaxa Basic Fibroblasts Nucleofector Kit (Lonza; Köln, Germany) with the nucleofector program U-023. Transfections with scrambled siRNA (ON-TARGETplus Non-targeting pool) served as controls. Transfected cells were seeded into 6-well plates and harvested at 48 h post-transfection for real-time PCR analysis.
Amaxa basic fibroblasts nucleofector kit
Manufactured by Lonza
Sourced in Germany
The Amaxa Basic Fibroblasts Nucleofector Kit is a laboratory equipment product designed for the transfection of primary human fibroblasts. It provides the necessary reagents and protocol for efficient nucleofection, a specialized electroporation-based method for delivering genetic material into these cells.
Automatically generated - may contain errors
Lab products found in correlation
On targetplus smartpool, by Horizon Discovery (3 mentions)
Nucleofector 2 transfection device, by Lonza (3 mentions)
On targetplus non targeting pool, by Horizon Discovery (2 mentions)
Nucleofector 2, by Lonza (1 mentions)
Pgl4.23 luc2 minp, by Promega (1 mentions)
Kpni bglii, by New England Biolabs (1 mentions)
4 protocols using amaxa basic fibroblasts nucleofector kit
1
siRNA Knockdown of RARA and RXRA
2
Transient Knockdown of RXRA and THRB
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hTCF and hTMC were transiently transfected with specific siRNA (ON-TARGETplus SMARTpool; GE Healthcare Dharmacon, Freiburg, Germany) for RXRA (150 pmol; target sequences: GCGCCAUCGUCCUCUUUAA, GCAAGGACCGGAACGAGAA, AGACCUACGUGGAGGCAAA and UCAAAUGCCUGGAACAUCU) or THRB (300 pmol; target sequences: UGGAAGUGUUCGAGGAUUA, GAGAAGAAAUGUAAAGGGU, GGACAAGCACCAAUAGUCA and CGAAAUCAGUGCCAGGAAU) by electroporation using the Nucleofector II transfection device (Lonza) and the Amaxa Basic Fibroblasts Nucleofector Kit (Lonza) with the nucleofector program U-023. Transfections with scrambled siRNA (ON-TARGETplus Non-targeting pool; GE Healthcare Dharmacon) were served as controls. Transfected cells were seeded into 6-well plates in duplicate and harvested at 48 h post-transfection for real-time PCR analysis.
3
Evaluating Genetic Variant Enhancer Activity
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Two different reporter vectors, the pGL4.23 [luc2/minP] (Promega, Madison, WI), which contains a gene unspecific minimal promoter sequence (TATA-Box, initiator element, RNA polymerase binding site), and the pGL4.10-LOXL1 reporter vector containing the specific LOXL1 core promoter sequence (extending from −1438 bp to the ATG start codon), which was cloned as previously described (17 (link)), were used. To test for enhancer activity, an intronic region of 200 bp surrounding rs7173049 (with either A/A or G/G genotype at the polymorphic site) was PCR-amplified with specific primers (Supplementary Material, Table S1 ) using genomic DNA extracted from human blood samples. The amplified products were cloned into pGL4.10-LOXL1 and pGL4.23 [luc2/minP] vectors via Kpn-I/Bgl-II (New England Biolabs, Ipswich, MA) double digestion and confirmed by sequencing.
For dual luciferase reporter assays hTCF and hTMC were transiently transfected by electroporation using the Nucleofector II transfection device (Lonza, Köln, Germany) and the Amaxa Basic Fibroblasts Nucleofector kit (Lonza) as previously described (17 (link)). Depending on the experiment, 5.5 μg of pGL4.10 or pGL4.23 reporter constructs were used together with 0.9 μg Renilla luciferase plasmid pGL4.74 (Promega). Data represent at least three independent experiments for each cell type.
For dual luciferase reporter assays hTCF and hTMC were transiently transfected by electroporation using the Nucleofector II transfection device (Lonza, Köln, Germany) and the Amaxa Basic Fibroblasts Nucleofector kit (Lonza) as previously described (17 (link)). Depending on the experiment, 5.5 μg of pGL4.10 or pGL4.23 reporter constructs were used together with 0.9 μg Renilla luciferase plasmid pGL4.74 (Promega). Data represent at least three independent experiments for each cell type.
4
Transient siRNA Knockdown of UPF1 and RXRα
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hTCF (0.75 × 106) were transiently transfected with specific siRNA (ON-TARGETplus SMARTpool, GE Healthcare Dharmacon, Freiburg, Germany) for UPF1 (75 pmol; target sequences: CAGCGGAUCGUGUGAAGAA, CAAGGUCCCUGAUAAUUAU, GCAGCCACAUUGUAAAUCA, GCUCGCAGACUCUCACUUU) or RXRα (150 pmol; target sequences: GCGCCAUCGUCCUCUUUAA, GCAAGGACCGGAACGAGAA, AGACCUACGUGGAGGCAAA, UCAAAUGCCUGGAACAUCU) by electroporation using the Nucleofector II transfection device (Lonza) and the Amaxa Basic Fibroblasts Nucleofector Kit (Lonza) with the nucleofector programme U-023. Transfections with scrambled siRNA (ON-TARGETplus Non-targeting pool, GE Healthcare Dharmacon) served as controls. Transfected cells were seeded into six-well plates in duplicate and collected at 48 h post transfection for real-time PCR analysis.
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