Topreal qpcr 2x premix sybr green with low rox

Total RNA from the spinal cord was isolated using TRIzol reagent according to the manufacturer’s protocol (GeneAll, RoboEx^TM, Thermo Fisher Scientific, Waltham, MA, USA). RNA was quantified using the NanoDrop spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared from total RNA using a kit (Enzynomics, Daejeon, Korea). Quantitative polymerase chain reaction (qPCR) was performed using the AriaMax Real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) with the TOPreal™ qPCR 2X premix (SYBR Green with low ROX; Enzynomics). The primers used for rat GAPDH, TNF-α, IL-1β, and COX2 were as follows: rGAPDH 5′-CTCATGACCACAGTCCATGC-3′, and antisense 5′-TTCAGCTCTGGGATGACCTT-CT-3′; rTNF-α 5′-AGATGTGGAACTGGCAGAGG-3′, and antisense 5′-CCCATTTGGGAACTTCTC-CT-3′; rIL-1β, 5′-CAGCAGCATCTCGACAAGAG-3′, and antisense 5′-CATCATC-CCACGAGTCACAG-3′; rCOX2 5′-CAGTATCAGAACCGCATTGCC-3′, and antisense 5′-GAGCAAGTCCGTGTTCAAGGA-3′.

According to the manufacturer’s instructions, total RNA was extracted from liver tissues (approximately 100 mg) using GENEzol™ Reagent (Geneaid, Taipei, Taiwan). Using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), the RNA concentration and purity were assessed. cDNA was synthesized using TOPscriptTM RT DryMIX (dT18/dN6) (Enzynomics, Daejeon, Republic of Korea) following the manufacturer’s instructions. Primers’ sequences for inflammatory markers are shown in Table 1.
The PCR reactions contained 10 µL of TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Republic of Korea), 1 µL of cDNA, 1µL of each primer, and RNase-free water in a total volume of 20 µL. PCR was conducted using the following thermal cycler protocol: Initial denaturation by pre-incubation at 95 °C for 12 min, cDNA was amplified for 40 cycles of denaturation at 95 °C for 15 s, annealing for 30 s as mentioned in Table 1, and extension at 72 °C for 30 s. PCR amplification and analysis were achieved using a CFX96™ Real-Time System (BIO-RAD, Hercules, CA, USA). The target gene’s critical threshold (Ct) was used to calculate the fold change using the 2^−∆∆Ct method. The housekeeping gene GAPDH was used to normalize the data.

Total RNAs were extracted from the pine samples under the same conditions as the in vivo transcriptome analysis. First-strand cDNA was synthesised by using SuperiorScript™ III cDNA Synthesis Kit (Enzynomics, Daejeon, Korea) with oligo-dT primers in accordance with the manufacturer’s protocol. Several genes belonging to major systems in co-expression patterns were subjected to qPCR with specific primers (Table S1). qPCR analysis was performed with TOPreal™ qPCR 2X PreMix (SYBR Green with low ROX) (Enzynomics) on a Rotor-gene Q (Qiagen, Valencia, CA, USA). Relative expression levels were calculated using the comparative 2^−ΔΔCT method with elongation factor (PITA_47202) as the internal control. The qPCR results are shown as the means ± SD of three independent biological replicates.

Total RNAs in cells and tissues were isolated using TRIzol™ (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed using AccuPower CycleScript RT Premix (Bioneer, Daejeon, Korea) and 1 µg RNA in a thermocycler. Real-time PCR amplification was performed using 96-well optical plates, with TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Daejeon, Korea), 400 nM each of forward and reverse primer and 1 µL of cDNA and H₂O in a 20 µL reaction volume. Real-time fluorescence of PCR products was detected using CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA) at following thermocycling conditions: 1 cycle of 95 °C for 3 min; 39 cycles of 95 °C for 10 s, and 60 °C for 30 s; 1 cycle of 95 °C for 10 s and 65 °C for 5 s followed by 0.5 °C increments at 5 s/step back to 95 °C. Only primer pairs leading to the synthesis of a single fragment with the appropriate size were used in this study. Primer sets used for this study were listed in Table S1. The relative gene expression was calculated using a 2^−∆∆ct method, which was normalized by β-actin expression level.