Sybr green pcr master mix reagent

The expression profiles of 13 randomly selected genes were confirmed through qRT-PCR (Table S1). The RNA of the kernels was extracted using TRIzol reagent according to the manufacturer’s protocol (Tiangen, Beijing, China). The Primer-BLAST online NCBI tool was used to design primers specific to each DEG. The cDNAs were synthesized using a FastKing RT kit (Tiangen, Beijing, China) and qRT-PCR was performed following the protocol of the SYBR Green PCR Master Mix Reagent (SuperReal PreMix Plus, Tiangen, China) on an ABI 7500 fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Seita.7G294000 (Actin 7) was used as the internal reference (Qin et al., 2020 (link); Yu et al., 2020 (link)) and the 2^−∆∆Ct method was used to calculate the relative expression level of each assessed gene (Livak & Schmittgen, 2001 (link)).

Tissue materials were harvested as previously mentioned. TRizol reagent (Tiangen, Beijing, China) was used to extract total RNA from the tissues of soybean and Arabidopsis. A miRcute miRNA first-strand cDNA synthesis kit (Tiangen) was used to perform first-strand cDNA synthesis of gma-miR172. QuantScript RT Kit (Tiangen) was used to perform cDNA synthesis of other genes. The corresponding mature miRNA sequence was applied as sense primers and antisense adaptor primers were provided in the SYBR Green PCR Master Mix Reagent (miRcute miRNA qPCR Detection Kit, Tiangen). A Chromo4 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) was used for performing the Real-time quantitative RT-PCR. Relative abundance of mRNA and miRNA were determined by qRT-PCR according to the method of 2^−ΔΔCt based on C_t values [39 (link)]. Actin4 gene was used as an endogenous control for soybean and Actin8 gene was used for an endogenous control in Arabidopsis. The sense primer for qRT-PCR of mature gma-miR172a was 5′-AGAATCTTGATGATGCTGCAT-3′. The primers used in real time RT-PCR analyses, including Actin4, Actin8, Glyma03g33470 and flowering control genes were shown in Table 2.

To validate the reliability of gene expression generated by the RNA-seq, 7 DEGs were randomly selected and tested. Briefly, the total RNAs were isolated from the foxtail millet leaves using the TRIzol reagent (Tiangen, Beijing, China) according to the manufacturer’s instructions. A FastKing RT kit (Tiangen, Beijing, China) was employed to synthesize the first-strand cDNA. A quantitative real time PCR (qRT-PCR) was performed using the SYBR Green PCR Master Mix Reagent (SuperReal PreMix Plus, Tiangen, China) on an ABI 7500 (Applied Biosystems, Foster City, CA, USA). The changes in gene expression were determined by the 2^−ΔΔCt method [63 (link)]. The foxtail millet Actin 7 (AF288226.1) gene was used as a reference gene. Three biological and three technical replicates were run for each gene. The primers for qRT-PCR used in the gene expression analysis were listed in Table S3.