Cfx connect rt qpcr system

Total cellular RNA was extracted with TRIzol reagent (Takara, Japan) and then reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, Japan). RT-qPCR was performed in a CFX Connect™ RT-qPCR System (Bio-Rad, USA) using Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). Pre-denaturation was conducted for 5 min at 95°C, followed by cycling with denaturation at 95°C for 10 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, repeated for a total of 40 cycles. Up to 40 cycles without results were counted as the maximum of 50 cycles. The relative expression values of six genes in different cell lines were calculated using the method of 2^^-ΔΔCt, with GAPDH and PBMC used as reference, respectively. The experiments were repeated three times to obtain the data. All primer sequences, synthesized by Sangon Biotech (Shanghai, China), are shown in Supplementary Table 2.

N. pseudonarcissus cDNA for bulbs, roots, stems, leaves, and flowers was generated from 1 to 2 μg of RNA using the Qiagen omniscript RT kit according to manufacturer’s protocol (QIAGEN, Germany). The experiment was performed in triplicate. A total reaction volume of 20 μL containing 1x SensiFAST SYBR Lo-ROX mix, 200 μM of each forward and reverse primers (Additional file 4) and cDNA sample was used for RT-qPCR analysis. Histone was used as internal reference gene. Real-time quantitative PCR was performed on CFX Connect RT-qPCR System from Bio-rad (USA). PCR conditions for amplification were 95 °C for 3 mins, 95 °C for 10 s, annealing temperature 52 °C for 30 s for 40 cycles. This was followed by dissociation step (as provided by software) - 95 °C for 10 s, 65 °C for 5 s and 95 °C for 5 s. The amplification efficiency was determined at 92% and a melting curve analysis confirmed NpNBS PCR product specificity. Norbelladine synthase relative expression values were determined by comparative 2^-ΔΔCt method and were scaled to lowest ddCq value by dividing ddCq of a sample with a minimum ddCq value identified among the samples [39 (link)].

Total RNA from AML cells was isolated using TRIzol reagent (#9108, Takara, Japan), then reverse transcribed into cDNA with PrimeScript RT regent Kits (#RR600, Takara, Japan). RT-qPCR was carried out with SYBR® Premix Ex Taq™ II kits (#RR820A, Takara) in a CFX Connect™ RT-qPCR System (Bio-Rad, USA). All primers were synthesised by Sangon Biotech (Shanghai, China). Primer sequences are given in Table S1. The number of cycles at which the fluorescence intensity increment reaches a threshold in each sample tube was taken as the Ct value. The Ct values of the gene to be tested and the internal reference gene were read separately for each specimen. Using β-actin as the internal reference, the relative expression of the gene to be tested was further calculated for each specimen using 2^-ΔΔCt23 (link).