Accuprep rna extraction kit

Jurkat cells were pretreated for 3 h with A.S. extract alone or combined with axitinib or dovitinib, followed by a 1 h incubation with PMA (50 ng/mL) plus PHA (1 μg/mL) in culture media (5% FBS). Total RNA was isolated using the AccuPrep^® RNA Extraction Kit (Bioneer Corp., Daejeon, Korea), and cDNA was synthesized from 100 ng total RNA using the iScript^TM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time RT-PCR was performed using iQ™ SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA) and the CFX96^TM Real-Time PCR System (Bio-Rad, Sacramento, CA, USA). Cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The following primers were used: IL-2 forward, 5′-ACTTTCACTTAAGACCCAGGGA-3′ and reverse, 5′-AGTGTTGAGATGATGCTTTGACA-3′; GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-GATCTCGCTCCTGGAAGATG-3′. All reactions were performed in triplicate, and the data were analyzed using the 2^−ΔΔCT method [39 (link)].

Cells were incubated in 60 mm² dishes for 24 h. Total RNA was isolated using the AccuPrep® RNA Extraction Kit (Bioneer Corp., Korea) and cDNA synthesized from 1 μg of total RNA using oligo (dT) primers (Bioneer Corp., Korea) and the RocketScript^TM Reverse Transcriptase Kit (Bioneer Corp., Korea). qRT-PCR was performed with the ExcelTaq 2X Q-PCR Master Mix (SMOBiO, Taiwan) and CFX96^TM Real-Time System (Bio-Rad, USA). The cycling conditions were as follows: 95°C for 3 min followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The primer sequences used in this study are provided in S1 Table. All reactions were run in triplicate, and data were analyzed using the 2^−ΔΔC_T method [37 (link)]. The internal standard was GAPDH. Significance was determined by the Student’s t-test with GAPDH-normalized 2^−ΔΔC_T values [37 (link)].

Total RNA was isolated from the frozen buffy coat specimens of human MM patients using the NucleoSpin^® RNA Blood extraction kit (Macherey-Nagel, GmbH&Co. KG, Germany). RPMI8226 cells were treated with IL-6 (10 ng/mL) in RPMI-1640 medium containing 0.1% FBS for 0 - 60 min. After incubation, the total RNA was isolated from RPMI8226 cells using the AccuPrep® RNA Extraction Kit (Bioneer Corp., Daejeon, Korea) and the cDNA synthesized from 1 μg of total RNA using oligo (dT) primers (Bioneer Corp., Daejeon, Korea) and the RocketScript™ Reverse Transcriptase Kit (Bioneer Corp., Daejeon, Korea). Quantitative real-time RT-PCR was performed using ExcelTaq 2X Q-PCR Master Mix (SMOBiO, Hsinchu, Taiwan) and the CFX96™ Real-Time PCR System (Bio-Rad, Sacramento, CA, USA). The cycling conditions were as follows: 95°C for 3 min followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Table 1 lists the target-specific primer sequences. All reactions were performed in triplicate with GAPDH as an internal standard. The data were analyzed using the 2^−ΔΔC_T method [29 (link)].