Screenfect sirna

Small interfering RNA for RXFP1 was purchased from Takara Bio (Shiga, Japan). The target sequences of siRNA against RXFP1 were as follows: siRNA-1, 5′-GGACUGAAUAGCCUUACUATT-3′ (sense), and 3′-TT CCUGACUUAUCGGAAUGAU-5′ (anti-sense); and siRNA-2, 5′-GCAGAACAAUUACAGUUC UTT-3′ (sense), and 3′-TT CGUCUUGUUAAUGUCAAGA-5′ (anti-sense). In addition, medium GC duplex #2 (Invitrogen, Carlsbad, CA, USA) was also used as a negative control (siC). siRNA, 10 nM, was transfected into cells (1 × 10⁵ cells/mL) using ScreenFect siRNA (Wako Purechemical Industries, Osaka, Japan) for 72 h. The procedure for the ScreenFect siRNA-mediated transfection in a 48-well culture plate was as follows. The siRNA was diluted in 16 μL of buffer, was mixed with 16 μL of buffer containing 0.8 μL ScreenFect siRNA, and was then incubated for 30 min at 25 °C before being added to each well.

Hepa1-6 were transfected with Atf4 siRNA (30 nmol/L) (SASI_Mm02_00316863, Merck KGaA, Darmstadt, Germany) and Non-Targeting siRNA (scramble) (MISSION^® siRNA Universal Negative Control, Merck) in EMEM, using ScreenFect™siRNA (FUJIFILM Wako Chemicals, Ltd. Osaka, Japan) according to the manufacturer’s protocol. Cells were incubated with transfection reagent for 24 h before gene expression analysis.

We transfected cells with siRNA oligonucleotides in Screen Fect Dilution Buffer (FUJIFILM Wako Pure Chemical Corporation) using Screen Fect siRNA (FUJIFILM Wako Pure Chemical Corporation), according to the manufacturer’s protocol. SiRNA sequences were as follows: RPL11 siRNA#1, 5′-GGUGCGGGAGUAUGAGUUA-3; RPL11 siRNA#2, 5′-CAAAUAAAUUCCCGUUUCUAUCC-3; scramble siRNA, 5′-UUCUCCGAACGUGUCACGU-3′.