Genmute sirna transfection kit

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.

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.