Rocketscript reverse transcriptase kit

BMDCs (2 × 10⁶ cells/well) were treated for 24 h with 14BME20 or LPS. Total RNA from the BMDCs was extracted with RiboEX total RNA kit (GeneAll Biotechnology, Seoul, Korea) and reverse transcribed into cDNA using a RocketScript™ Reverse Transcriptase kit (Bioneer Corporation, Daejeon, Korea). After cDNA was synthesized, the cDNA was amplified by PCR using AccuPower® PCR PreMix (Bioneer Corporation). The sequences of the RT-PCR primers used in this study were as follows: murine TGF-β forward, 5′-TAT AGC AAC AAT TCC TGG CG-3′ and reverse, 5′-TCC TAA AGT CAA TGT ACA GC-3′; murine IL-10 forward, 5′-AGA AAT CAA GGA GCA TTT GA-3′ and reverse, 5′-CTG CAG GTG TTT TAG CTT TT-3′; murine IDO forward, 5′-TTA TGC AGA CTG TGT CCT GGC AAA CTG-3′ and reverse, 5′-TTT CCA GCC AGA CAG ATA TAT GCG GAG-3′; murine COX-2 forward, 5′-GTG GAA AAA CCT CGT CCA GA-3′ and reverse, 5′-TGA TGG TGG CTG TTT TGG TA-3′; murine IL-12p40 forward, 5′-TTA TGC AAA TTG TGA GCT TG-3′ and reverse, 5′-CCT TTG CAT TGG ACT TCG GTA G-3′; murine β-actin forward, 5′-CGC AGA GTC TCG CCA TTA TG-3′ and reverse, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′. After cDNA amplification, the products were separated on 1.5% (w/v) agarose gels and stained with StainingSTAR (DyneBio, Gyeonggi-do, Korea). The relative expression of cytokines was analyzed by Image J software.

Total RNA was extracted using Ribospin™ II (GeneAll^®, Seoul, Republic of Korea; 314-103) by the manufacturer’s instructions. cDNA was synthesized from 1 µg total RNA by RocketScript™ Reverse Transcriptase Kit (Bioneer, Daejeon, Republic of Korea; E-3141). RT-PCR was conducted using amfiXpand PCR Master Mix (GenDEPOT, Katy, TX, USA; P0331-050) by the manufacturer’s instructions. The whole thermos-cycle processes were performed in Veriti™ 96-Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA; #9902), The used primers are listed on Table 1.

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)].