Sybr green real master mix

Real-time quantitative PCR was performed with a LightCycler 96 Real-Time PCR System (Roche) using SYBR Green Real Master Mix (Tiangen). General cycling conditions were set to: incubation at 95 °C for 3 min, followed by 40 cycles for 10 s at 95 °C, 30 s at 62 °C and 20 s at 72 °C. The comparison between the internal reference gene 40S ribosomal protein S11 and β-actin by BestKeeper gave decreasing ranking order for S11 and β-actin (http://150.216.56.64/referencegene.php?type=reference) [46 (link)]. Expression of mRNA was normalized with the expression of 40S ribosomal protein S11 in each sample. The Relative expression was determined using the 2^(-ΔΔCt) method [39 (link), 47 (link)]. The primers for Ccpgrp5 and Ccpgrp6 are shown in Tables 1 and 2 respectively. The efficiencies of each primers for Ccpgrp5, Ccpgrp6 and internal reference gene S11 were 95.78, 100.67 and 99.43% respectively. Each PCR was performed with triplicate samples.

Human breast cancer cells included MDA-MB-231 cells, MDA-MB-231 luc cells, SUM-159 cells, and SUM-159 luc cells (provided by Xi'an Medical University) were incubated at 37°C with 5% CO₂ in RPMI-1640 (GIBCO) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific), penicillin (100 IU/ml), and streptomycin (100 mg/ml). Cells were passaged three times a week. The Britanin working solutions (10 mM Britanin dissolved in DMSO) provided by Shanghai Jiaotong University were prepared by dilution of the stock solution in fresh culture medium on the day of use. Britanin, as a natural product, was purified by high-performance liquid chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy. The purity of Britanin was greater than 95% (Figure S1). A Cell Counting Kit-8 (Dojindo), Hoechst staining kit (Beyotime), TUNEL Apoptosis Detection kit (Beyotime), D-Luciferin potassium salt (Sciencelight), and antibodies (Abcam) were used in this study. Total RNA RNA extraction and CDNA synthesis used RNAiso Plus and the Reverse Transcription System (TaKaRa, Tokyo, Japan). Quantitative RT-PCR (qRT-PCR) analysis was performed in a 7300 Real-Time System (ABI, New York, America) using the SYBR Green RealMasterMix (TIANGEN, Beijing, China).

Total RNA was isolated from soybean seedlings and hairy roots, or from Arabidopsis seedlings using Trizol reagent (TaKaRa, Japan). The first strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) based on the manufacturer’s instructions. qRT-PCR was performed on ABI prism 7500 Real-Time PCR system (Applied Biosystem, United States) using SYBR Green Real Master Mix (Tiangen, China) with the following PCR cycles: 95°C for 15 min, followed by 40 cycles of amplification (95°C for 10 s, 58°C for 20 s, and 72°C for 32 s). The data of qRT-PCR were determined using the 2^-ΔΔCt method according to the cycle threshold (Ct) values (Livak and Schmittgen, 2001 (link)). The soybean CYP2 (GmCYP2) (Glyma.12g024700) and Arabidopsis actin2 (AtACT2) (At3g18780) genes were used for normalization. Student’s t-test was used to determine significant differences. A level of 0.05 was used for statistical significance. Primer sequences for qRT-PCR analysis were designed using the software tool Primer Premier 5.0. The primer sequences are listed in Supplementary Table S1. All reactions were conducted with four biological replicates for each sample.

Quantitative PCR was applied to determine the CNVs of 11 loci in ECAY fragments from the 283 male samples mentioned above, which consisted of 217 Chinese indigenous horses, 65 foreign horses, and a Przewalski’s horse. The primers used for qPCR are shown in Supplementary Table S3. GAPDH, which has two copies on autosomes, was used as a reference gene for normalization. On each plate, we set up wells for standard curve samples, a calibrator, and a negative control. All the test samples were run in triplicate to ensure accuracy. If CT values of same sample differ by 0.3, this sample needed to retest. qPCRs for amplifying were performed in a 20 μl reaction mixture including 10 μl of SYBR Green RealMaster Mix (TIANGEN BIOTECH, Beijing), 0.6 μl of primers (10 pmol/μl), 7.8 μl ddH20, and 1 μl of DNA template (5 ng/μl). Amplifications were conducted in a CFX96 Real-Time System thermal cycler (Bio-Rad, United States). The PCR thermal cycling conditions were as follows: 95°C for 15 min, followed by 39 cycles at 95°C for 10 s, annealing at 56–60°C for 20 s, 72°C for 30 s, and the melting curve was then generated by taking fluorescent measurements every 0.5°C from 65°C until 95°C. The CNVs were determined with the methods described by Hamilton et al. (2009) (link).

Experiments in this section were performed routinely in triplicate with control and nicotine induction cultures of P. putida S16 harboring pME6032, pME-Gad, or pME-GirrE. A single colony of recombinants or control was picked randomly from MSM plate, and a 1:100 dilution of a fresh overnight culture was inoculated in three 250-mL flasks containing 50 mL MSM (control) or MSM plus 1 g/L nicotine (induction).
Batch cultures were incubated at 30°C with shaking (200 rpm) to early-exponential phase (OD₆₀₀ ∼0.5). The early-exponential cells were harvested by centrifugation at 14,000 × g for 2 min. Total RNA was extracted from ∼1 × 10⁸ cells using an RNAprep pure cell/bacteria kit (Tiangen), and quantified by NanoDrop (Thermo Fisher Scientific). Then, total RNA was treated by 0.8 mg of DNase (Fermentas), and reverse transcribed to cDNA using FastKing cDNA Kit (Tiangen). The cDNA was diluted 1:10 and served as the template for qPCR analysis using the CFX96 Real-Time PCR Detection system (Bio-Rad) with SYBR Green RealMasterMix (Tiangen) and qPCR primers (Tang et al., 2013 (link)). The threshold cycle (C_T) values for each target gene were normalized with the reference 16S rRNA gene. The 2^ΔΔCT method was used to calculate the relative expression level, where ΔΔC_T = (C_T^target − C_T^16S)_induction − (C_T^target − C_T^16S)_control (Livak and Schmittgen, 2001 (link)).

Real-time PCR was performed in triplicate for each sample on an iQ5 Real-time PCR instrument (Bio-Rad) using SYBR Green Real Master Mix (TIANGEN, China). 40S ribosomal protein S11 gene served as an internal reference gene to normalize the mRNA expression in different tissues as previous studies described (Engelsma et al. 2001 (link)). The primers that were used have been shown in Table 1. Reaction conditions were as follows: incubated for 1 min at 94 °C, followed by 40 cycles of 20 s at 94 °C, 20 s at 59 °C and 50 s at 70 °C. The PCR data were analyzed using 2^(−ΔΔCt) method.

The inducible expression profiles of ccIRF5, ccTNFα and two ISG genes [ccISG15 and ccPKR (protein kinase R)] upon poly I:C and LPS challenges were analysed by real-time PCR using gene-specific primers (Table 1) on an iQ5 Real-time PCR instrument (Bio-Rad). Reactions were performed in a 20 μl volume using SYBR Green RealMasterMix (TIANGEN). Cycling conditions were one cycle of 94 °C for 5 min, followed by 40 cycles of 94 °C for 20 s, 60 °C for 30 s and 72 °C for 20 s. The 40S ribosomal protein S11 gene was amplified in parallel as a housekeeping control for normalization [27 (link)]. The amplification efficiency of the primers used in the real-time PCR was between 0.80 and 0.86. All samples were analysed in triplicate. Standard curves were run on the same plate. The real-time PCR data were analysed with the 2^-△△CT method.