Sybr premix ex taqtm perfect real time

Gene transcript levels were analyzed using Q-PCR with a BIO-RAD CFX Connect^TM Optics Module (Bio-Rad, USA). The cDNA was synthesized from RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Actin was used as an internal control in gardenia (forward primer: 5′-GCGAGGAAACAAGTGGAAGACTA-3′; reverse primer: 5′-TGCCAACCACCATTTATTAGGAG-3′)^{29 (link)}. All gene-specific primers in this study were shown in Supplemental Table S1, and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Q-PCR was performed using the SYBR^® Premix Ex Taq^TM (Perfect Real Time) (TaKaRa, Japan) and contained 12.5 µL 2 × SYBR Premix Ex Taq^TM, 2 µL cDNA solution, 2 µL mix solution of target gene primers and 8.5 µL ddH₂O in a final volume of 25 µL. The amplification was carried out under the following conditions: 50 °C for 2 min followed by an initial denaturation step at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, 50 °C for 15 s, and 72 °C for 30 s. Gene relative expression levels of target genes were calculated by the 2^−△△Ct comparative threshold cycle (Ct) method^{68 (link)}. The Ct values of the triplicate reactions were gathered using the Bio-Rad CFX Manager V1.6.541.1028 software.

qRT-PCR was used to detect gene expression levels with a BIO-RAD CFX Connect^TM Optics Module (Bio-Rad, Des Plaines, IL, USA), and their values were calculated referring to the 2^−△△Ct comparative threshold cycle (Ct) method [51 (link)]. The cDNA was synthesized from RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Tokyo, Japan). qRT-PCR was performed using the SYBR^® Premix Ex Taq^TM (Perfect Real Time) (TaKaRa, Tokyo, Japan) and contained 12.5 mm³ 2 × SYBR Premix Ex Taq^TM, 2 mm³ cDNA solution, 2 mm³ mix solution of target gene primers and 8.5 mm³ ddH₂O in a final volume of 25 mm³. The amplification was carried out under the following conditions: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, 52 °C for 30 s and 72 °C for 30 s. All used primers were listed in Table S2.

Total RNA of each group were extracted respectively using Trizol solution (Invitrogen) at 24 h after transfection. Fast-strand cDNA was generated from 1 μg of total RNA using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan). Real-time qPCR was performed in an ABI PRISM 7500 Real-Time System. A 10-fold dilution of each cDNA was amplified in a 50 μl volume, using the SYBR Premix Ex TaqTM Perfect Real Time (TaKaRa, Shiga, Japan). The primers used were as follows: JMJD2A: forward 5′-ATCCCAGTGCTAGGATAATGACC-3′, reverse 5′-ACTCTTTTGGAGGAACCCTTG-3′; ARHI: forward 5′-GATTACCGCGTCGTGGTAGTC-3′, reverse 5′-TCAATGGTCGGCAGGTACTCA-3′; GAPDH: forward, 5′-TGACGCTGGGGCTGGCATTG-3′, reverse 5′-GCTCTTGCTGGGGCTGGTGG-3′.
Primers were synthesized by Shanghai Daweike Biotechnology Co. Ltd (Shanghai, China). PCR cycle conditions were 95°C for 30 s, and 40 cycles of 95°C for 5 s and 60°C for 34 s. The amplification specificity was evaluated with melting curve analysis. Relative mRNA was determined by using the formula 2^-ΔCT (CT, cycle threshold) where, as described previously [40 (link)]:
$\mathrm{\Delta CT}=\mathrm{CT}\left(\mathrm{target}\mathrm{gene}\right)-\mathrm{CT}\left(\mathrm{GAPDH}\right).$