Cells were seeded into p35 dishes and simultaneously transfected with 5 μl of 10 μM duplex siRNAs (sequences given in table S1) (TriFECTa Kit DsiRNA Duplex, Integrated DNA Technologies) using Opti-MEM (Invitrogen) and Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. The following day, cells were split and seeded into two glass-bottom microscopy dishes in the medium indicated above for live cell imaging experiments and transfected again with siRNA using the same protocol. After 8 hours, around 30% of the growth medium was exchanged, and the cells were transfected with the plasmids encoding fluorescent proteins as indicated above.
Trifecta kit dsirna duplex
Manufactured by Integrated DNA Technologies
Sourced in United States
The TriFECTa Kit DsiRNA Duplex is a laboratory product manufactured by Integrated DNA Technologies. It is designed to provide Dicer-substrate short interfering RNA (DsiRNA) duplexes for gene silencing applications. The kit includes the necessary components for the formation of DsiRNA duplexes, which can be used to investigate gene function and expression.
Automatically generated - may contain errors
6 protocols using trifecta kit dsirna duplex
1
Transient Transfection of Cells
2
TSPO Knockdown and TNF Response Evaluation
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Cells were plated onto 12-well plates at an initial concentration of 3×105 cells per well, and immediately transfected using the TriFECTa® kit DsiRNA duplex (Integrated DNA Technologies, Inc., Coralville, IA, USA) using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific).
The following small-interfering (si)RNA duplexes (50 nM) used for TSPO (NM_000714) were: duplex1,5- CGA CCA CAC UCA ACU ACU GCG UATG-3 ; duplex 2,5-ACG CUU UCA UGA CCA CUG GGC CUG C -3 ; and duplex 3,5-UAC GGC UCC UAC CUG GUC UGG AAA G -3 . Gene expression and target gene knockdown were evaluated by RTqPCR. After 48 hours, cells were treated (or not) with TNF at different time points. In these different conditions, mock and scrambled were used as controls.
The following small-interfering (si)RNA duplexes (50 nM) used for TSPO (NM_000714) were: duplex
3
Silencing LINC00885 in Breast Cancer Cells
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LINC00885 expression was silenced in T47D and MCF7 cells using the TriFECTa Kit DsiRNA Duplex (Integrated DNA Technologies, Coralville, IA, USA). Three siRNA duplexes targeting LINC00885 transcript were used: siRNA-1 (5′-UAAGAAAUCCCCAUGAUUCCACUGGGG-3′), siRNA-2 (5′-UCCUCAGGUGUUGGUGCUUUAUCUUGG-3′) and siRNA-3 (5′-GAAUCAGAUUAGAUCCAUUCUGCUCAG-3′). A siRNA without target in human transcriptome was included (5′-AUACGCGUAUUAUACGCGAUUAACGAC-3′) as non-target control. Transfection of siRNA duplexes (150 pmol/well) were performed with Lipofectamine 3000 in 6 well plates at 70% confluence cells and incubated overnight. Cell culture medium was replaced by a siRNA free culture medium and cells were incubated 48 h.
4
MMAB Gene Silencing Protocol
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Three siRNAs (siMMAB-1, siMMAB-2, and siMMAB-3) targeting MMAB were purchased from TriFECTa® Kit DsiRNA Duplex (Integrated DNA Technologies, USA). The sequences of these siRNAs were GCA GGA AAG ACA GGU AAA GUG AUT G, AGU ACC UUC ACA GGA GAA AGG AGA C, and GGC GGA CAA GAU UUG GAA GUU UAG T, respectively. Optimal siRNA sequence was confirmed by qRT-PCR and western blot. The qRT-PCR primer & probe information are listed in Supplementary Table S1 .
5
TIGAR Silencing Modulates Redox Homeostasis
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Two ICC cell lines, HuCCT1 and SSP‐25 (Riken Cell Bank), were cultured in RPMI medium (Thermo Fisher Scientific) supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. Negative control and TIGAR siRNA (TriFECTa Kit DsiRNA Duplex) were purchased from Integrated DNA Technology and transfected 48 h after seeding cells by forward transfection using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. Cellular ROS assay kit, glycolysis assay, NADP/NADPH assay, and oxidized glutathione (GSSG)/reduced glutathione (GSH) quantification kits were purchased from Dojindo (cat. 340‐09811, 343‐09921, 344‐09331, and 342‐09011, respectively; Kumamoto, Japan). A malondialdehyde (MDA) assay kit was purchased from JaICA (cat. KMD‐008W). A liproxstatin and caspase‐3/7 fluorescence assay kit was purchased from Cayman‐Chemical (cat. 950455‐15‐9 and cat. 10009135). CDDP was purchased from Sigma‐Aldrich (cat. 15663‐27‐1).
6
Silencing H19 and HIF-2α in Cells
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Small interference to H19 and to HIF-2α was obtained with TriFECTa Kit DsiRNA Duplex (Integrated DNA Technologies) transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions and as in the work of [72 (link)]. Scrambled universal negative control RNA duplex (NC1) was used. RNA was extracted after 48 h and chromatin was crosslinked after 18 h from transfection.
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