Rt qpcr primers

Total RNA was extracted from cells with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using the TOYOBO ReverTra Ace qPCR RT kit, according to the manufacturer's protocol. qPCR was performed using the KAPA SYBR-Green Supermix PCR kit (Kapa Biosystems). RT-qPCR primers were obtained from Sangon Biotech Co., Ltd.; sequences are listed in Table II. The reaction was started at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 61°C for 30 sec, and 72°C for 30 sec. Relative gene expression levels were measured using the cycle threshold values and the 2^−ΔΔCq method (20 (link)).

Total RNA was isolated from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was then performed. The RT-qPCR primers for RAC1 and GAPDH (internal control) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RT was performed using a FastQuant RT kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. The PCR primers for RAC1 and GAPDH were as follows: RAC1, forward 5′-ATGTCCGTGCAAAGTGGTATC-3′, reverse 5′-CTCGGATCGCTTCGTCAAACA-3′; and GAPDH, forward 5′-GCCAAAAGGGTCATCATCTC-3′ and reverse 5′-GTAGAGGCAGGGATGATGTTC-3′. qPCR was performed using Taq PCR MasterMix (Tiangen Biotech Co., Ltd.) and a ViiA™ system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermal cycling conditions were as follows: Initial denaturation at 95°C for 60 sec, 40 cycles of amplification at 95°C for 20 sec, annealing and extension at 60°C for 30 sec. GAPDH was used as an internal control for PCR amplification. The data were analyzed using the 2^−ΔΔCt method (25 (link)).

TransScript II All-in-One First-Strand cDNA Synthesis SuperMix for PCR and TransScript II SYBR-Green Two-Step RT-qPCR SuperMix kit (AH321-01 and AQ301-01; both from Transgen Biotech Co., Ltd.); RT-qPCR primers [Sangon Biotech (Shanghai) Co., Ltd.]; Trizol kit (10296028; Thermo Fisher Scientific, Inc.); RIPA lysis buffer, BCA kit and ECL chromogenic reagent (P0013C, P0012S, and P0018FS; all from Beyotime Institute of Biotechnology); monoclonal rabbit anti-rat CaMKII, monoclonal rabbit anti-rat Cx43, and monoclonal rabbit anti-rat β-actin antibodies, as well as polyclonal horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (ab5683, ab79010, ab179467, and ab6728; all from Abcam); KN93 (CSN11255; CSNpharm).

Total RNA was extracted from cultured HepG2 cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was eluted with RNasefree water and the concentration was measured by UV detection at 260 nm. Subsequently, cDNA was synthesized using the Transcriptor First-Strand cDNA Synthesis kit (Takara Bio, Inc.). qPCR was performed with SYBR-Green Master Mix (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The RT-qPCR primers were purchased from Sangon Biotech Co., Ltd. The fold-change for mRNA relative to β-actin was determined using the 2^-∆∆Cq method (19 (link)). The specific primer sequences were as follows: CYP4A11 (human) forward, 5′-ACG GCT TGC TCC TGT TGA AT GG-3′; CYP4A11 reverse, 5′-AGA GGT CAG GCT GTA GAT GGT GTC-3′; IL-6 (human) forward, 5′-CAC ACA GAC AGC CAC TCA CC-3′; IL-6 reverse, 5′-AGT GCC TCT TTG CTG CTT TC-3′; TNF-α (human) forward, 5′-AAC CTC CTC TCT GCC ATC AA-3′; TNF-α reverse, 5′-CTG AGT CGG TCA CCC TTC TC-3′; IL-1β forward, 5′-GGA CAA GCT GAG GAA GAT GC-3′; IL-1β reverse, 5′-TCG TTA TCC CAT GTG TCG AA-3′; β-actin forward, 5′-TTG CTG ACA GGA TGC AGA A-3′; and β-actin reverse, 5′-ACC AAT CCA CAC AGA GTA CTT-3′.