Real time pcr kit

Total RNA was isolated from leaves or hairy roots of soybean using Trizol reagent (Invitrogen, Shanghai, China), and RT-qPCR analysis was performed on a LightCycler96 instrument (Roche, Switzerland) with a real-time PCR kit (TOYOBO, Osaka, Japan). The gene expression levels were calculated by the 2^−ΔΔCt method with GmEF1β (GenBank accession no. NM_001248778) and GmTUB4 (GenBank accession no. EV263740) as the internal control. The reaction conditions consisted of an initial 5 min pre-incubation at 94 °C, and 40 cycles at 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 40 s, followed by a melting-curve analysis from 55 °C to 100 °C with a final cooling for 10 min at 72 °C. The relative transcript abundance of each target gene was calculated using the 2^−∆∆CT method. The primers used for expression analysis are shown in Table S2.

Total RNA was isolated from ‘Williams’ and ‘L77-1863’ soybean leaves using Trizol reagent (Invitrogen, Shanghai, China). cDNA synthesis was conducted using an M-MLV reverse transcriptase kit (Takara, Dalian, China) according to the manufacturer’s instructions. RT-PCR was performed to analyse GmPIB1 transcript levels in ‘Williams’ and ‘L77-1863’ plants according to Zhang et al. (2012) (link). The soybean housekeeping gene GmEF1β (GenBank accession no. NM_001248778) was used as the internal control. qRT-PCR analysis was performed to measure GmPIB1 transcript levels on a CFX96 Touch™ Real-Time PCR machine (Bio-Rad, USA) using a real-time PCR kit (Toyobo, Japan). The soybean housekeeping gene GmEF1β was used as an internal reference to normalize all data. The relative transcript level of the target gene was calculated using the 2^−ΔΔCT method. Three biological replications per line were performed in each test.

Quantitative real-time PCR analysis was performed to determine the transcript abundance of GmIFR. Total RNA was isolated from “Suinong 10” soybean leaves using Trlzol reagent (Invitrogen, Shanghai, China). The synthesis of cDNA was conducted using an oligo(dT) primer and a M-MLV reverse transcriptase kit (Takara, Dalian, China) according to the manufacturer's instructions. qRT-PCR was performed on a CFX96 Touch™ Real-Time PCR machine (Biobad, USA) using the real-time PCR kit (ToYoBo, Japan). DNA accumulation was measured using SYBR Green as the reference dye. The soybean housekeeping gene Gmactin4 (GenBank accession no. AF049106) was used as the internal control (see Supplementary Table S1 for primer sequences). For tissue distribution analysis, the transcript level of GmEF1 gene (GenBank accession no. NM_001248778) was used as quantitative control (see Supplementary Table S1 for primer sequence). The relative expression of target gene in different tissues of soybean was calculated using the 2^−ΔΔCT method. For each sample, three biological replicates were analyzed with their respective technical replicates.