Sybr green real time pcr master mix

RNA samples from the 12 animals used for the RNA-seq experiment were analyzed by qPCR. Total cDNA was synthesized using reverse transcriptase Kit (TaKaRa, Dalian). QPCR were performed using LightCycler 480II Real-Time PCR System and SYBR^® Green PCR Master Mix (TaKaRa, Dalian). Each real-time RT-PCR reaction (in 25 μL) involved 12.5 μL 2 × SYBR Green Realtime PCR Master Mix (TaKaRa, Dalian), 1 μL of each primer, 2 μL cDNA and 8.5 μL H₂O. The cycling conditions included an initial single cycle (95 °C for 3 min), and followed by 40 cycles (95 °C for 15 s; 57 °C for 15 s; 72 °C for 20 s). All amplifications were followed by dissociation curve analysis of the amplified products. Specific primers were designed using the NCBI, specificities were confirmed with BLAST and gene expression levels were normalized with RPS20 to attain the relative expression by using 2 ^(−ΔΔCt) value methods (Table 1). The statistical difference in gene expression was analyzed by SAS during different endometrium development stages of pregnancy. The correlation between the results of RNA-seq and qPCR was calculated using correlation test.

PAMs were collected at 6 h, 12 h, 24 h, 36 h and 48 h post-infection. The total RNA from all groups was extracted using Trizol and reverse transcribed using a PrimeScript RT reagent kit (TaKaRa Shuzo, Tokyo, Japan). A relative quantification of the porcine immune molecules was carried out using the corresponding primers.
All RT-qPCR analysis was performed using a 7500 Fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). The RT-qPCR reaction mixture consisted of 10 µL of 2 × SYBR Green Real time PCR Master Mix (TaKaRa, Tokyo, Japan), 2 µL of cDNA, 1 µL of each primer (0.4 µM) and 6 µL Rnase free water. The following program was used for RT-qPCR assays: polymerase activation at 95 °C for 20 s, followed by 40 cycles for melting at 95 °C for 15 s and extension at 60 °C for 30 s. Relative quantification of the expression of target genes for all groups was calculated using the 2^−ΔΔct method. All samples were tested in triplicate, and the RT-qPCR primers used in this study are listed in Table 2.

To validate RNA-seq data, the expression levels of 12 genes from multiple groups were quantified by quantitative PCR (qPCR) using 2^−ΔΔCt value methods. Ileum total RNA used for RNA-seq was performed to synthesize cDNA using reverse transcriptase Kit (TaKaRa, Dalian, China). Specific primers of these genes were designed using NCBI database, porcine GAPDH gene was designed as an endogenous control (Table S1). The qPCR reactions were performed in 20 μL system involved in 9.5 μL 2 × SYBR Green Realtime PCR Master Mix (TaKaRa, Dalian), 1 μL of forward and reverse primers, 1 μL cDNA and 7.5 μL RNase free ddH₂O using LightCycler 480 II Real-Time PCR System. The cycling conditions included an initial denaturation (95°C for 3 min), followed by 30 cycles (95°C for 15 s; 60 ± 1°C for 15 s; 72°C for 20 s). Three independent biological replicates were performed in triplicate.

Total RNA was isolated from soybean tissues using TRIzol reagent (Invitrogen) and treated with DNase I (Invitrogen) to avoid genomic DNA contamination. First-strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). Gene-specific primers were designed according to gene sequences using Primer 5.0 software (Table S1). The quantitative RT-PCR was performed with a CFX96TM real-time system (Bio-Rad) in a 20 μl system containing 2 μl of a tenfold diluted cDNA, 10 μl of 2 × SYBR green real-time PCR master mix (Takara), and 1 μl each of 10 μM forward and reverse primers. β-actin was used as the internal control. Statistical analyses were performed using the t-test, and p < 0.05 and < 0.01 were considered significant and extremely significant differences, respectively.

Total RNA was extracted from cells using a TRIzol kit (ThermoFisher Scientific, Waltham, MA, USA). A PrimeScript RT reagent kit (Takara, Kyoto, Japan) was used to synthesize complementary DNA. Next, qRT-PCR was performed to assess the mRNA expression of ANXA6 using 2 × SYBR Green real-time PCR Master Mix (Takara) and an Applied Biosystems 7300 Real Time PCR system (Applied Biosystems). The 2^−ΔΔCt method was used to quantify the data, and β-actin was used as a reference gene in the analysis. The forward primer was 5′-ACGGTTGATTGTGGGCCTG-3′, and the reverse primer was 5′-GTGCATCTGCTCATTGGTCC-3′.

The mRNA expression pattern of the FADS2 transcript in various tissues (kidney, skin, muscle, gill, spleen, eye, stomach, intestine, blood, brain and heart) and liver of Japanese seabass fed different diets were measured by real-time fluorescent quantitative PCR (qRT-PCR) using β-actin (GeneBank accession number: HE577671.1) as reference gene. Specific primers for FADS2 (FADS2-RTF and FADS2-RTR) and β-actin (β-actin-F and β-actin-R) were designed using Primer 5.0 software based on the partial cDNA sequences obtained as described previously (Table 3). The real-time PCR was carried out in a quantitative thermal cycler (Mastercycler eprealplex, Eppendorf, German) in a final volume of 25 µl containing 2×SYBR Green Real-time PCR Master Mix (TaKaRa, Japan), primer pairs and cDNA. The program was 95°C for 30 s followed by 35 cycles of 95°C for 5 s, 58°C for 15 s and 72°C for 20 s. Melting curve (1.85°C increment/min from 58°C to 95°C) was performed after the amplification phase for confirmation. Each sample was run in triplicate. The gene expression levels of putative FADS2 in liver of Japanese seabass fed different diets were studied by qRT-PCR method: 2^−ΔΔCT[38] (link).

For RNA isolation and qRT-PCR, the leaves, stems, roots and pollen of P. tomentosa were harvested, immediately frozen in liquid nitrogen and stored at –80 °C for further analysis. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with on-column treatment using RNase-free DNase I (Qiagen) according to the manufacturer’s instructions to ensure no genomic DNA contamination. First-strand cDNA was synthesized with approximately 1 µg of purified total RNA using the SuperScript III reverse transcription kit (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed in the LightCycler 480 Detection System (Roche, Penzberg, Germany) with two PtoActin genes as internal reference. The details of the primers used are listed in Table S2. The reaction mixture (20 µL) contained 10 µL 2× SYBR Green Real-time PCR Master Mix (TaKaRa, Dalian, China), 0.5 µM of each of the forward and reverse primers, and 2 µL of cDNA template. The amplification was completed with the following cycling parameters: 95 °C for 30 s; followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s; 60 °C for 60 s and 50 °C for 30 s. qRT-PCR was carried out in triplicates (technical repeats) to ensure the reproducibility of the results. The relative expression ratios were calculated from the threshold cycle according to the delta-delta CT method [47 (link)].