Sybr premix ex taq 2 mix

Total cellular RNA was extracted with RNAiso Plus (TaKaRa). Random hexamer primers were used to synthesize cDNA using a Transcriptor First Strand cDNA synthesis kit (Roche), and SYBR Premix Ex Taq II mix (TaKaRa) was used for real-time PCR (RT-PCR). Reactions were carried out in triplicate on an ABI StepOnePlus system (Applied Biosystems). The relative levels of expression of mRNA were calculated using threshold cycle (ΔΔC_T) analysis, and the values were normalized to the relative mRNA expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primers were used: 5′-CCATTCTGATTTGCTGCCTTAT-3′ and 5′-TTTCCTTGCTAACTGCTTTCAGTA-3′ for CXCL10, 5′-CTGTGCGAGTGTACCGGATG-3′ and 5′-ATCCCCACATGACTTCCTCTT-3′ for IRF1, and 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-3′ for GAPDH.

Total RNA was extracted from the colon samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocols. RNA quantification was performed using a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA). cDNA was then synthesized using a first-strand cDNA synthesis kit (Thermo Scientific, USA) and 2 μg total RNA. qRT-PCR was subsequently conducted using the QuantStudio™ 6 Flex Real-Time PCR System (ABI, USA) and SYBR® Premix Ex Taq™ II mix (Takara, Japan). The gene-specific primer pairs used are listed in Table 3. The 2^−ΔΔCT method was used to determine the relative mRNA levels.

Quantitative real-time PCR (qRT-PCR) was conducted to quantify gene expressions. The RNA samples used for qRT-PCR were the same as with those used for the transcriptome sequencing. The experiments were performed on Applied Biosystems 7500 Fast Real-Time PCR platform with the SYBR^® Premix Ex Taq™ II mix (Takara Biotechnology Co., Ltd, Dalian, Japan), and the results were analyzed by the Applied Biosystems 7500 software (Applied Biosystems Life Technologies). The results of triplicate assays were then calculated by the 2^-ΔΔCt method. The primers for qRT-PCR analysis are listed in Table S2.

The plants were processed with the following treatments in the experiment to analyze the gene expression patterns in soybean plants (G. max) [86 (link)]. In order to determine the dose-dependent expression patterns, the seedings were cultivated in a solution with 0, 50, 100, 150, and 200 mM NaCl for 8 h. The plant materials were collected after 0, 2, 4, 8, 12, and 24 h in the 200 mM NaCl treatment to determine the time-dependent expression patterns. To determine organ-specific expression, the plant organs stored in the −80 °C refrigerator described in Section 4.1 were used for qPCR identification [31 (link)].
The total RNA was extracted from the plant samples using the RNA-easy Isolation Reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions and then reverse transcribed into cDNA using PrimeScript RT kit (Takara, Shiga, Japan). The gene expression level was evaluated by qPCR using the 2^−ΔΔct method using real-time PCR on a CFX96™ Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA) with SYBR Premix ExTaq™ II Mix (TaKaRa, Shiga, Japan) [86 (link)]. GmACTIN6 (GeneBnak Accession: AAK285830.1) was used as an internal control for three technical replicates. All primers were designed using the NCBI Primer tool (http://www.ncbi.nlm.nih.gov/, accessed on 4 February 2022) and are shown in Table S2.

Total RNA, cDNA, and primers were obtained as described above. The amplicon specificity was verified by melting curve analysis (Figure S7) and agarose gel electrophoresis. The qRT-PCR was performed on a Roche 480 Realtime detection system (Roche Diagnostics, Rotkreuz, Switzerland) following the manufacturer’s instructions, using SYBR Premix ExTaq II Mix (TaKaRa, Dalian, China) in a final volume of 15 μL containing 2 μL cDNA, 7.5 μL SYBR Premix ExTaq II (TaKaRa, Dalian, China), and 200 nM of forward and reverse primers. The amplification program was set as follows: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 20 s, and extension at 72 °C for 20 s. The experiments were performed in triplicate. The amplification efficiencies (E) of primer pairs were estimated by qRT-PCR using 1×, 5×, 10×, 20×, and 30× dilutions of cDNA, according to the equation: E = 10^−1/slope−1 [55 (link)]. Primers and amplification efficiencies of qRT-PCR were shown in Table S4. GmUKN1 was used as the reference gene [56 (link)], and the relatively constant Ct values of GmUKN1 across all samples demonstrated its invariant expression under our experimental conditions (Table S5), suggesting that it is suitable for the internal control of qRT-PCR. The relative expression levels of test genes were analyzed by the 2^−△△Ct methods [57 (link)].

Total RNA was extracted from uterine tissues with RNAiso Plus solution (TaKaRa, China). Then total RNA samples were reverse-transcribed into single-stranded cDNA in a 25 μL reaction mixture (TaKaRa, China). Quantitative PCR was performed using a SYBR Premix Ex TaqIIMix according to manufacturer’s protocol (Takara, China). The results were terminally analyzed through ABI Prism 7500 software (Applied Biosystems, USA) and housekeeping 18s mRNA level was regarded as an internal control. The primers were as follows: 18s, 5’-AATCAGGGTTCGATTCCGGA-3’ (sense) and 5’-CCAAGATCCAACTACGAG CT-3’ (antisense); prl8a2, 5’- TTATGGGTGCATGGATCACTC-3’ (sense) and 5’-CCCACGTAAGGTCATCATGGA-3’ (antisense). Melting curve analysis and agarose gel electrophoresis were performed after the quantitative PCR assays to supervise the purity of PCR products.