A small interfering RNA (siRNA) duplex targeting TCTP (5′-GAAATCAATCAAAGGGAAA-3′) and a nonsilencing control siRNA duplex were synthesized by RIBOBIO (Guangzhou, China). A TCTP expression plasmid and a control plasmid were purchased from Origene (Beijing, China). Cells were cultured in antibiotic-free medium for 2 hours. They were then transfected with TCTP siRNA (50 nM) or TCTP plasmid DNA (2.5 ng/ul) using Lipofectamine 2000 (Invitrogen, USA). Silencing was evaluated 40 hours after transfection by western blot. TCTP overexpression was evaluated 24 hours after transfection by western blot.
Sirna duplex
Manufactured by RiboBio
Sourced in China
SiRNA duplex is a synthetic double-stranded RNA molecule designed to mediate RNA interference (RNAi) and silence the expression of a specific target gene. It functions by binding to and inducing the degradation of the target mRNA, thereby reducing the production of the corresponding protein.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Sirna transfection reagent, by RiboBio (2 mentions)
Scrambled sirna, by RiboBio (2 mentions)
Hdac2, by RiboBio (1 mentions)
Hdac3 sirna duplex, by RiboBio (1 mentions)
Hdac1, by RiboBio (1 mentions)
Sodium phosphate, by Merck Group (1 mentions)
Pluronic f68, by Merck Group (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Cholesterol, by Avanti Polar Lipids (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n methoxy polyethylene glycol 2000 dspe peg2000, by Avanti Polar Lipids (1 mentions)
1 2 distearoyl sn glycero 3 phosphatidylcholine dspc, by Avanti Polar Lipids (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Matrigel matrix, by BD (1 mentions)
6 protocols using sirna duplex
1
Modulating TCTP Expression via siRNA
2
Modulating hiPSC-CM Contractility via siRNA
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A small interfering RNA (siRNA) duplex against PLN or ANK2 was designed and constructed by Ribobio (Guangdong, China). Next, hiPSC-CMs were transfected with siRNA at a final concentration of 100 nM using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction. A non-matching siRNA was used as a negative control. After siRNA transfection for 48 h, the cells were then incubated with SNT (3 µM) for 24 h. Cellular contractility was then determined by CardioExcyte 96 apparatus.
3
Targeting HDAC1-3 Modulates p75NTR and Histone Acetylation
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HDAC1, HDAC2, and HDAC3 siRNA duplex (Guangzhou RIBOBIO CO, LTD) were used to interfere with endogenous HDAC1, HDAC2 and HDAC3 mRNA levels, respectively. Cells were seeded in six-well culture microplates at a density of 1 × 105 cells/well in 2 ml of antibiotic-free normal growth medium. The following day, cells were transfected with 50 nM siRNA duplex and 12 μl of siRNA transfection reagent (Guangzhou RIBOBIO CO, LTD) mixed in DMEM/F12 (1:1) media with 1% fetal bovine serum. The transfected cells were incubated at 37°C for 24 h. Untreated cells and non-specific siRNA (scrambled siRNA; Guangzhou RIBOBIO CO, LTD) were used as controls. The interference efficiency was evaluated by qRT-PCR and western blot analyses. In addition, the expression levels of p75NTR mRNA and protein, and Ace-H3K9 and Ace-H4K12 levels were investigated by the above methods. This experiment was performed in duplicate and repeated three times.
4
Lipid-based siRNA Delivery System
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1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[meth-oxy(polyethyleneglycol)-2000] (DSPEPEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[meth-oxy (polyethyleneglycol)-2000-folate (DSPE-PEG2000-folate)], and cholesterol were purchased from Avanti Polar Lipids, China. Pluronic F68, calcium chloride, and sodium phosphate were purchased from Sigma-Aldrich. A siRNA duplex was designed by Guangzhou RiboBio Co., Ltd. (Guangzhou, People’s Republic of China). It is a 21 bp double-stranded RNA oligos with dTdT 3′ overhangs and has sequences as follows: (sense) 5′-GGACAUAUGAGACCUUCAAdTdT-3′; and (antisense) 5′ UUGAAGGUCUCAUAUGUCCdTdT-3′.
5
Silencing HDAC1, 2, and 3 in SH-SY5Y cells
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HDAC1, 2, and 3 small-interfering RNA (siRNA) duplexes (Guangzhou RiboBio Co, Ltd, China) were used simultaneously (si-HDAC1-2-3) or singly (si-HDACs) to interfere with endogenous HDAC1, HDAC2, and HDAC3 expression. The siRNA oligos were as follows: HDAC1: 5′-CCGGTCATGTCCAAAGTAA-3′; HDAC2: 5′-TCCGTAATGTTGCTCGATG-3′; and HDAC3: 5′-GCATTGATGACCAGAGTTA-3′. In brief, differentiated SH-SY5Y cells were seeded in 6-well culture microplates at a density of 1 × 105 cells/well in 2 mL of antibiotic-free normal growth medium. The following day, cells were transfected with a solution of 50 nmol/L siRNA duplex and 12 μL of siRNA transfection reagent (Guangzhou RiboBio Co, Ltd) mixed in DMEM/F12 medium containing 10% FBS. The transfected cells were incubated at 37°C for 24 hours. The untreated (non-siRNA) and nonspecific siRNA treated (scrambled siRNA; Guangzhou RiboBio Co, Ltd) cells were used as controls. The interference efficiency was evaluated by qRT-PCR and Western blot analyses. In addition, the expression levels of TrkA mRNA and protein were investigated by the above methods. This experiment was performed in duplicate and repeated 3 times.
6
HEK293 and hiPSC-CM Co-Transfection Protocol
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Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (Gibco) with 5% CO2 at 37°C. The HEK293 cells were transiently co‐transfected with Kv4.3 (or Kv4.2), KChIP2, and DPP6 plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The green fluorescent protein (GFP) was used as a reporter. The human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs, 50‐60 days of maturity; Cellapybio, Beijing, China) were maintained in serum‐free medium (Cellapybio) on 24‐well dishes coated with Matrigel Matrix (BD‐Biocoat, Corning, USA) with 5% CO2 at 37°C. The small interfering RNA (siRNA) duplex against the human DPP6 gene (NM_130797, 5′‐GGTCCATCATCGGCTCTTT‐3′) was designed and constructed by Ribobio (Guangdong, China). hiPSC‐CMs were transfected with siRNA using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction at a final siRNA concentration of 100 nM. A non‐matching siRNA was used as a negative control. Cells were used for experiments 48 hours after transfection.
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