Real time rt pcr kit

Quantitative real-time PCR analysis was performed using a real-time RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions, on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, USA). Total RNA was extracted from soybean leaves using Trizol reagent (Invitrogen, Shanghai, China) and 1 μg was converted to first-strand cDNA using an M-MLV reverse transcriptase kit (Takara, Dalian, China). Amplification was performed using the primer pair GmERF113-qF and GmERF113-qR (Supplementary Table 1). For tissue distribution analysis, the transcript levels of the GmEF1β gene (GenBank accession no. NM_001248778) were used as an internal control (Supplementary Table 1 for primer sequences). The soybean housekeeping gene GmActin4 (GenBank accession no. AF049106) was used as an internal control (see Supplementary Table 1 for primer sequences) for treatments with abiotic and biotic stresses. Relative expression levels were calculated using the 2^-ΔΔCt method. qRT-PCR analysis was performed using three biological replicates (i.e., RNA samples extracted from three independent plants) and three technical replicates of each biological replicate.

RNA-seq data were further validated using qRT-PCR for six selected genes, using gene-specific primer sets (Fig 3A). Primer pairs were designed using the Primer 5 software. Actin was amplified along with the target gene as an endogenous control to normalize expression between different samples. qRT-PCR was performed using a real-time RT-PCR kit (Takara, Japan), on a CFX96 Real-Time System (BioRad, USA). The delta-delta-cycle threshold (Ct) method was used to calculate the relative expression of each mRNA [37 ].

Candidate genes located within the 100-kb LD segment near a peak SNP were identified. Biosynthesis of unsaturated FAs of soybean was used for FA-related gene screening (https://www.kegg.jp/). The expression level of each FA-related gene was determined by real-time reverse transcription polymerase chain reaction (RT-PCR) analyses. Real-time RT-PCR amplifications were performed using the real-time RT-PCR kit according to the manufacturer’s instructions (Takara, Japan) on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). About 1 μg of total RNA was used for each reverse transcription. Each amplification reaction was performed with 1 μL of the resulting cDNA first-strand cDNA synthesis solution, 0.2 μM of each primer, and 10 μL of SYBR Green PCR Master Mix, in a total reaction of 20 μL. The PCR cycling conditions were as follows: 95 °C for 5 s, 60 °C for 20 s, 72 °C for 20 s for 40 cycles and 60 °C for 1 min. The relative mRNA level of each candidate gene was evaluated against soybean actin4 (GmACTIN) (GenBank Accession Number AF049106) reference gene in three replicates. The sequences of the primer pairs used in amplifying the candidate genes are presented in Additional file 6: Table S3.