HCT116 cells were transfected with four HOTAIR small interfering RNAs (siRNAs) (50 nM) using DharmaFECT reagent (Life Technologies) according to the manufacturer’s instructions. The following sequences were used: #1, 5′-GAACGGGAGUACAGAGAGAUU-3′; #2, 5′-CCACAUGAACGCCCAGAGA UU-3′; #3, 5′-UAACAAGACCAGAGAG CUGUU-3′; #4, 5′-GAGGAA AAGGGAAAAUCUAUU-3′; si-green fluorescent protein (siGFP), 5′-CUACAACAGCCACAACGUCdTd-3′.
Dharmafect reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
DharmaFECT is a transfection reagent used to deliver nucleic acids, such as siRNA, miRNA, and plasmid DNA, into a variety of cell types for gene expression and gene silencing studies. It is formulated to facilitate efficient and gentle delivery of nucleic acids into the cell.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
On targetplus smart pool human nfe2l2, by Horizon Discovery (1 mentions)
Nanofectin, by GE Healthcare (1 mentions)
Dharmacon on targetplus smartpool sirnas, by Horizon Discovery (1 mentions)
C jun sirna, by Santa Cruz Biotechnology (1 mentions)
C myb sirna, by Santa Cruz Biotechnology (1 mentions)
Ty 82, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
X tremegene 9 transfection reagent, by Roche (1 mentions)
19 protocols using dharmafect reagent
1
HOTAIR siRNA Knockdown in HCT116 Cells
2
Colorectal Cancer Cell Manipulation
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Patients and cell culture. Twenty patients with colorectal cancer were selected. Patients who had received chemotherapy or a family history of polypus or IBD before surgery were excluded. All samples were fixed in neutral buffered formalin 10% and paraffin blocked. All cut sections from both tumor and adjacent normal colorectal mucosa were selected, and immunohistochemical staining was performed for expression analyses. All patients who participated in the present study provided written informed consent. The experimental protocol for human subjects was approved by the Ethics Committee of The Second Affiliated Hospital of Guilin Medical University.
The human colorectal adenocarcinoma SW620 cells and normal colon cell line FHC were incubated in complete Leibovitz's L-15 medium with tetracycline-free 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), 100 U/ ml penicillin and 100 µg/ml streptomycin. SW620 cells were transfected with PARP6 siRNA or survivin siRNA using DharmaFECT reagent (Life Technologies, Grand Island, NY, uSA) according to the manufacturer's instructions. The siRNA sequences are listed in Table I.
The human colorectal adenocarcinoma SW620 cells and normal colon cell line FHC were incubated in complete Leibovitz's L-15 medium with tetracycline-free 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), 100 U/ ml penicillin and 100 µg/ml streptomycin. SW620 cells were transfected with PARP6 siRNA or survivin siRNA using DharmaFECT reagent (Life Technologies, Grand Island, NY, uSA) according to the manufacturer's instructions. The siRNA sequences are listed in Table I.
3
Inhibition of p38 Attenuates TGF-β1-Induced Fibrosis
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The involvement of p38 in the TGF-β1-induced expression of CTGF, collagen I and collagen III was further examined using the siRNA-mediated knockdown of p38. For transfection with p38 siRNA or non-targeting negative control (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), DharmaFECT reagent (Invitrogen Life Technologies) was used according to the manufacturer's instructions. After 24 h, the culture medium was replaced with fresh supplemented medium, and the cells were cultivated for an additional 24 h following treatment with 3 ng/ml TGF-β1 for 6 h. The expression levels of p38 and p-p38 were detected by immunofluorescence staining, and the transfection efficiency over time (0, 12, 24, 36 h) was also validated by RT-PCR (data not shown). The mRNA expression levels of CTGF, collagen I, and collagen III were detected by RT-qPCR. The protein expression of CTGF was detected by western blot analysis.
4
Targeted Gene Silencing by siRNA
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Specific small interfering RNA (siRNA) against ADAM17 (si-ADAM17) and MMP21 (siMMP21) and siRNA scrambled control (si-NC), were purchased from RIBBIO (Shanghai, China). Huh7 and SMMC7721 cells were transfected with plasmids or oligonucleotides using DharmaFECT Reagent (Invitrogen, Carlsbad, CA, USA). The sequences for siADAM17, siMMP21,siNC were as follows: siADAM17: 5′-GCTTGTTCATCGAGTGAAA-3′, 5′-GGATGGTCTAGCAGAATGT-3′; siMMP21: 5′-GATCCATAATGCAACCAAA-3′, 5′-ACTGGAAGGTAGTTAATGA-3′; siNC: TTCTCCGAACGTGTCACGTTT.
5
Silencing of GCN5 in glioma cells
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The glioma cell lines CHG5 and SHG44 were kindly provided by Xiuwu Bian from Third Military Medical University, China. U87 and U118 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). U251 cell line was obtained from China Center for Typical Culture Collection (CCTCC). Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (GEMINI, Woodland, CA, USA) and maintained in a humidified atmosphere at 37 °C with 5% CO2.
Two GCN5 siRNAs were synthesized from Invitrogen. Twenty-four hours prior to transfection, U87 and U251 cells were plated onto a 6-well plate at 70%–90% confluency. Cells were then transfected with siRNA at final concentration of 50 nM with the DharmaFECT reagent (Thermo Fisher Scientific, Lafayette, CO, USA) according to the manufacturer’s instrument. After 6 h of incubation at 37 °C, the transfection medium was replaced with 2 mL complete medium containing 10% FBS. Cells were collected for the following experiments at the indicated times.
Two GCN5 siRNAs were synthesized from Invitrogen. Twenty-four hours prior to transfection, U87 and U251 cells were plated onto a 6-well plate at 70%–90% confluency. Cells were then transfected with siRNA at final concentration of 50 nM with the DharmaFECT reagent (Thermo Fisher Scientific, Lafayette, CO, USA) according to the manufacturer’s instrument. After 6 h of incubation at 37 °C, the transfection medium was replaced with 2 mL complete medium containing 10% FBS. Cells were collected for the following experiments at the indicated times.
6
siRNA Transfection for MSI-1 and MSI-2
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One day before siRNA transfection, the cells were plated in six-well plates and incubated for 24 h at 37 °C to reach a confluency of 70–80%. The cells were either transfected with 10 nM each of MSI-1 (S8980) and MSI-2 siRNA (S42757) or negative control siRNA #1 (ThermoFisher Scientific, Waltham, MA, U.S.A.) in OPTI-MEM media (Life Technologies, Grand Island, NE, U.S.A.) via lipotransfection with Dharmafect® reagent (Thermo Fisher Scientific, Waltham, MA, U.S.A.). After 24 h of incubation, OPTI-MEM media were replaced by a DMEM culture medium. Then, 24 h after media change, the cells were used for further experiments.
7
Knockdown of MBD2 in MDA-MB-468 Cells
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A pre-validated human MBD2 siRNA constructs (catalogue ID numbers s17079 and s17080) and a control non-silencing, scrambled construct (catalogue number 4390846) were purchased from Thermo Fisher Scientific (Waltham, MA). MDA-MB-468 cells were transfected with these constructs using DharmaFECT reagent also from Thermo Fisher Scientific, following the manufacturer protocol.
8
NRF2 Gene Silencing Protocol
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Small interfering RNA experiments were performed by using human-specific NRF2 or non-targeting siRNA (On-TargetPlus SMART pool human NFE2L2; Dharmacon). Cells were transfected with 50 nM siRNA, using Dharmafect reagent (Thermo Scientific), according to the manufacturer's instructions.
9
Investigating HSPA8 Regulation in Endometrial Cancer
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The role of HSPA8 in endometrial carcinoma cells RL-95-2 and HEC-1B was examined using siRNA-mediated knockdown of HSPA8. For screening HSPA8 siRNA, 50 µM, 100 µM, and 200 µM HSPA8 siRNA (HSPA8-Homo-768: 5′-GCUGGUCUCAAUGUACUUATT-3′, 5′-UAAGUACAUUGAGACCAGCTT-3′; HSPA8-Homo-1112: 5′-GGCCAGUAUUGAGAUCGAUTT-3′, 5′-AUCGAUCUCAAUACUGGCCTT-3′; HSPA8-Homo-1509: 5′-GUCCUCAUCAAGCGUAAUATT-3′, 5′-UAUUACGCUUGAUGAGGACTT-3′) or nontargeting negative control (5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and DharmaFECT reagent (Thermo Fisher Scientific) were transfected into RL-95-2 cells according to the manufacturer’s instructions. After 48 hours, the expression of HSPA8 was confirmed by qRT-PCR, and the best HSPA8 siRNA was selected. Then, both RL-95-2 and HEC-1B cells were transfected with HSPA8 siRNA, the expression of HSPA8 was confirmed by Western blotting, and cell proliferation, cell cycle, and apoptosis were performed.
10
Optimizing RARB mRNA Silencing with siRNAs
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siRNAs were designed as 19mers with the help of eurofins mwg operon siRNA‐design tools (eurofins, Ebersberg, Germany). Three different siRNAs were chosen to cover different regions of the RARB mRNA, according to the pattern AA(N19) and with a GC‐content between 36 and 52%. Furthermore, one control siRNA with a Cy3‐tag was ordered, that does not bind to any mRNA within the cell. Sequences were siRARB1: UCAUCUGGAUAUCACUAUG, siRARB2: UUACCUCUUCACCAUCACA, siRARB3: CCAC UGACACCAACCUCAA, siControlCy3: AGGUAGUGU AAUCGCCUUGUU. DF‐1 cells (Himly et al., 1998 ) were cultured in DMEM medium containing 10% fetal calf serum and 1% penicillin, streptomycin and neomycin and maintained in a humidified 5% CO2/95% humidified air atmosphere at 37°C. The cells were transfected with the RARB siRNAs using either Dharmafect reagent (Thermo Fisher Scientific, Schwerte, Germany) or Nanofectin (PAA Laboratories, GE Healthcare, Freiburg, Germany) according to manufacturer's instructions.
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