A3R expression levels were knocked down with specific siRNAs at a final concentration of 60 nM (Invitrogen); negative siRNAs (Invitrogen) were used as controls. Before siRNA transfection, fresh Neurobasal media was added to cultured neurons plated for 10 days. The transfection cocktail containing 300 μl of transfection buffer (SignaGen), 12 μl of siRNA stock (15 μM) for each target protein, and 9 μl of GenMute™ reagent was added carefully to each dish along with 1 ml of media. After incubation (37°C, 5% CO2) for 5 h, the transfection media was replaced with fresh Neurobasal media, and neurons were treated with LDL cholesterol for 3 days. Knockdown efficiency was measured by immunoblotting as described below.
Transfection buffer
Manufactured by SignaGen
Sourced in United States
Transfection buffer is a laboratory reagent used to facilitate the introduction of genetic material, such as DNA or RNA, into cells. Its core function is to provide a suitable environment and conditions for the efficient delivery and uptake of the genetic material by the target cells.
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6 protocols using transfection buffer
1
Neuronal A3R Knockdown and LDL Treatment
2
siRNA Transfection in MC3T3-E1 and MDPC23 Cells
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The siRNAs, mimics and inhibitors were designed and synthesized by Guangzhou RiboBio Co., Ltd. (siRNA: Sense: 5′-GCUAGAACAGCAUGGUCCAdTdT-3′, antisense: 3′-dTdTCGAUCUUGUCGUACCAGGU-5′; inhibitors: 5′-UCCUCCCUCCCCUACCCGGUUCAAG-3′; mimic sense: 5′-AGGAGGGAGGGGAUGGGCCAAGUUC-3′, antisense: 5′-UCCUCCCUCCCCUACCCGGUUCAAG-3′) (Table I ). MC3T3-E1 and MDPC23 cells were seeded in 6-well plates at a density of 1×105/well. The cells were transfected with 20 nM siRNA mimics or inhibitors using GenMute™ siRNA Transfection kit and Transfection Buffer (SignaGen Laboratories) according to the manufacturer's protocol. To ensure a high silencing efficiency, the transfection medium with siRNAs was changed every 72 h. Experiments were performed 24–72 h post transfection.
3
Silencing of mmu_circRNA_003795 in BMSCs
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The small interfering (si)RNAs targeting mmu_circRNA_003795 were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences of siRNA were as follows: (Sense 5′-GCUAGAACAGCAUGGUCCAdTdT-3′ and anti-sense 3′-dTdTCGAUCUUGUCGUACCAGGU-5′). The target sequence corresponding to the sense and antisense siRNA oligonucleotides was as follows: 5′-GCTAGAACAGCATGGTCCA-3′. Negative control siRNA was designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China; the sequence is confidential). The mmu-miR-504-3p inhibitor was synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The sequence was as follows: 5′-AGGGAGAGCAGGGCAGGGUUUC-3′. BMSCs (1×105) were transfected with 20 nM siRNA or inhibitor using GenMute™ siRNA Transfection kit and Transfection Buffer (SignaGen Laboratories, Rockville, MD, USA) according to the manufacturer's protocol. Then the cells were incubated for 72 h at 37°C prior to the sequential tests.
4
TRPML1 Knockdown in Cultured Neurons
5
Targeted Knockdown of Neuronal Proteins
6
Silencing mmu_circRNA_007893 in RAW264.7 Macrophages
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The small interfering RNAs (siRNAs) targeting mmu_circRNA_007893 were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences of siRNA1-3 were showed as followed: siRNA1, 5′-GCCUUCAUCCAGUCCAAUGdTdT-3′ (sense) and 3′-dTdTCGGAAGUAGGUCAGGUUAC-5′ (antisense); siRNA2, 5′-CAGCUUGCCUUCAUCCAGUdTdT-3′ (sense), 3′-dTdTGUCGAACGGAAGUAGGUCA-5′ (antisense); and siRNA3, 5′-CCCUCCAGCUUGCCUUCAUdTdT-3′ (sense) and 3′-dTdTGGGAGGUCGAACGGAAGUA-5′ (antisense), respectively. Negative siRNA was used as a control. The RAW264.7 macrophages were transfected with siRNA using GenMute™ siRNA Transfection Kit and Transfection Buffer (SignaGen Laboratories, Rockville, MD, USA) according to the manufacturer's instructions. Then the cells were incubated for 48 h at 37°C prior to the sequential tests.
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