Stealth rnai sirna duplex oligoribonucleotides

HK-2 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well without antibiotics in the medium for 24 h. Orifice plates were divided into four groups: Con, LPS, siRNA and siRNA + LPS and three wells per group were used. Con and LPS-treated cells were incubated with medium, and siRNA and siRNA + LPS-treated cells were grown and transfected with 6 pM siRNA using Stealth RNAi siRNA Duplex Oligoribonucleotides and RNAiMAX ((Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were incubated for 36 h and then placed in D-MEM/F12 medium with LPS (50 μM) which was replaced in LPS and siRNA + LPS groups for 3 h and serum-free medium was used in Con and siRNA groups. To quantify NGAL and caspase 3 mRNA expression, RNA was harvested and cDNA was synthesized.

Target-specific siRNAs (Stealth RNAi siRNA Duplex Oligoribonucleotides, Thermo Fisher Scientific, Waltham, MA, USA) were transfected using Lipofectamine RNAiMAX (13778100, Thermo Fisher Scientific, Waltham, MA, USA). Stealth RNAi siRNA Negative Control, Low GC (12935110, Thermo Fisher Scientific, Waltham, MA, USA) was used as the control siRNA.
siTBK1-15′-GCGAGAUGUGGUGGGUGGAAUGAAU-3′siTBK1-25′-GGGAACCUCUGAAUACCAUAGGAUU-3′siFBXO22-15′-UCGUGUGGUCCUUGUCUUUGGUUAU-3′siFBXO22-25′-GCUGUAAGGUGGGAGCCAGUAAUUA-3′

Stealth RNAi™ siRNA Duplex Oligo ribonucleotides [Hmox1 RNAi (Thermo Fisher Scientific) and Stealth RNAi™ siRNA Negative Controls (Thermo Fisher Scientific)] for HO-1 and negative control small interfering RNAs (siRNA) were used for RNA interference. The sense and antisense strands of HO-1 siRNA were as follows:
sequence #1, 5′-CAGCUCUAUCGUGCUCGAAUGAACA-3′ (sense)
and 5′-UGUUCAUUCGAGCACGAUAGAGCUG-3′ (antisense);
sequence #2, 5′-GCUUUAAGCUGGUGAUGGCUUCCUU-3′ (sense)
and 5′-AAGGAAGCCAUCACCAGCUUAAAGC-3′ (antisense);
sequence #3, 5′-GGCAGUGGGAAUUUAUGCCAUGUAA-3′ (sense)
and 5′-UUACAUGGCAUAAAUUCCCACUGCC-3′ (antisense).