Sybr green qpcr mix

qPCR was designed based on consensus sequences of each alignment and performed using Rotor-gene Q (2plex on PC) instrument (QIAGEN; Hilden, Germany) with SYBR Green qPCR Mix (Biofact). The following primers were used for qPCR: RANK-forward, 5′-AGAAGACGGTGCTGGAGTCT-3′; RNAK-reverse, 5′-TAGGAGCAGTGAACCAGTCG-3′; TRAF6-forward, 5′-GCCCAGGCTGTTCATAATGT-3′; TRAF6-reverse, 5′-TCGCCCACGTACATACTCTG-3′; OSCAR-forward, 5′-CTGCTGGATACGGATCAGCTCCCCAGA-3′; OSCAR-reverse, 5′-CCAAGGAGCCAGAACCTTCGAAACT-3′; TRAP-forward, 5′-CAGTTGGCAGCAGCCAAGGAGGAC-3′; TRAP-reverse, 5′-TCCGRGCTCGGCGATGGACCAGA-3′; Blimp1-forward, 5′-TGCTTATCCCAGCACCCC-3′; Blimp1-reverse, 5′- CTTCAGGTTGGAGAGCTGACC -3′; c-fos-forward, 5′-AGAGCGGGAATGGTGAAGAC-3′; c-fos-reverse, 5′-GCTGCATAGAAGGAACCGGA-3′; NFATc1-forward, 5′- CAACGCCCTGACCACCGATAG -3′; NFATc1-reverse, 5′-GGGAAGTCAGAAGTGGGTGGA-3′; MafB-forward, 5′-AGTGTGGAGGACCGCTTCTCT-3′; MafB-reverse, 5′-CAGAAAGAACTCAGGAGAGGAGG-3′; IRF-8-forward, 5′AGACGAGGTTACGCTGTGC-3′; IRF-8-reverse, 5′- TCGGGGACAATTCGGTAAACT -3′. Data were normalized against the expression of GAPDH as an internal control.

qPCR analyses were conducted using the Rotor‐gene Q (2plex on PC) instrument (QIAGEN) with SYBR Green qPCR Mix (Biofact). The primers used for qPCR analyses were presented in Table 1. Data were normalized against the expression of GAPDH as an internal control.

Total RNA extraction was performed using TRIzol reagent (15596018; Invitrogen, Carlsbad, CA, USA) following to the manufacturer’s instructions. cDNA was generated using the WizScript cDNA Synthesis Kit (W2202; Wizbiosolutions, Seongnam, Korea) with oligo-dT primer. Quantitative polymerase chain reaction (qPCR) next was performed using a Step OnePlus RT-PCR system (Applied Biosystems, Foster City, CA, USA) with specific primers (Table 1) and SYBR Green qPCR Mix (DQ485; BioFACT, Daejeon, Korea). The cycle threshold (Ct) value was confirmed using Step One software version 2.3 (Applied Biosystems, USA), and the mRNA fold-change value was determined using the 2 (−ΔΔCt) method [20 (link)].