Silencer sirna construction kit

Small interfering RNA (siRNA) sequences targeting the coding regions of human TRPV1 mRNA (5′-AACCTATGTAATTCTCAC CTACATCCT-3′), human TRPC1 mRNA (5′-AAGCTTTTCTTG CTGGCGTGC-3′), human TRPM2 mRNA (5′-AAAGCCTCAGTT CGTGGATTCTT-3′), and human TRPM7 mRNA (5′-AAGAAC AAGCTATGCTTGATGCT-3′) were used. The oligonucleotide sequence used for synthesis of non-targeting siRNA is 5′-GGGTATACTAGTGAATTAG-3′ (forward) and 5′-CTAATTCACTAGTATACCC-3′ (reverse). To construct siRNA oligomers, the Silencer siRNA Construction Kit (Ambion, Life Technologies Corporation, Carlsbad, CA, USA) was used according to the manufacturer's protocol. Transfection of siRNAs at 100 nM for human TRPV1, human TRPC1, and human TRPM2 or 300 nM for human TRPM7 to HepG2 cells were carried out using Lipofectamine 2000. Cells were treated with siRNAs of human TRPV1 or human TRPC1 for 24 h and siRNAs of human TRPM2 or human TRPM7 for 48 h. They were then subjected to RT-PCR, western blotting, [Ca²⁺]_i measurements, intracellular ROS measurements, trypan blue exclusion assays, Hoechst33342/propidium iodide (PI) assays, caspase 3/7 activity assays, or assessment of intracellular cytochrome c levels, as indicated in the individual experiments.

PKCα (forward, 5′-AAT CGT ACC GTG ACG TGG AGC CCT GTC TC-3′), (reverse, 5′-AAG CTC CAC GTC ACG GTA CGA CCT GTC TC-3′), PKCδ (forward, 5′-AAC CTC ACT ACG CAT AGA CTG CCT GTC TC-3′), (reverse, 5′-AAC CTC ACT ACG CAT AGA CTG CCT GTC TC-3′), PLD1(forward, 5′-AAA GAG GTG GTT GAT AGT AAA CCT GTC TC-3′), (reverse, 5′-AAT TTA CTA TCA ACC ACC TCT CCT GTC TC-3′) specific small interfering RNA (siRNA) were prepared using the Silencer siRNA Construction kit (Ambion) according to manufacturer’s protocol. A nonspecific scrambled siRNA was generated with same GC content for control.

PKCδ (Sense 5′-AAC CTC ACT ACG CAT AGA CTG CCT GTC TC-3′), (Antisense 5′-AAC CTC ACT ACG CAT AGA CTG CCT GTC TC-3′), Sphk1 (Sense 5′-AAG AGC TGC AGA GCC TTG CCC CCT GTC TC-3′), (Antisense 5′-AAG GGC AAG GCT CTG CAG CTC CCT GTC TC-3′), Sphk2 (Sense 5′- AAG TCG CTG TAT GTG TAG GGC CCT GTC TC-3′), (Antisense 5′ AAG CCC TAC ACA TAC AGC GAC CCT GTC TC -3′) specific small interfering RNA (siRNA) were prepared using the Silencer siRNA Construction kit (Ambion) according to manufacturer's protocol. A nonspecific scrambled siRNA (control siRNA) was generated with same GC content for control.