Primescript rt regent kit with gdna eraser

TRIzol reagent (Takara, Shiga, Japan) was used to isolate total RNA samples based on instructions provided by the manufacturer. 1 ug of RNA was used to synthesize cDNA with PrimeScript^™ RT regent Kit with gDNA Eraser (Takara, Dalian, China). TB Green^® Premix Ex Taq™ (Takara, Shiga, Japan) and a StepOne Plus system (Applied Biosystems, CA, USA) were used to carry out qRT-PCR. The sequences of primers were listed in Table S2. The 2^-ΔΔCt method was used to calculate the relative expression of each gene.

RT-qPCR was used to analyze differences in the expression levels of amylase genes between the deletion mutant ∆POX01907 and the parental strain ∆PoxKu70 according to a previously described method [16 (link)]. Total RNA from both ∆POX01907 and ∆PoxKu70 was extracted and used as a template to generate first-strand cDNA for RT-PCR using the PrimeScript RT regent kit with gDNA Eraser (TaKaRa). Each qPCR comprised a 20-μL volume, including 2.0 μL of the template for first-stand cDNA, 10 μL of SYBR Premix ExTaq II, 0.8 μL of 10 μM primer (either forward or reverse), and 6.4 μL of sterile water, subjected to initial denaturation for 3 min at 98 °C, followed by 40 cycles of 10 s at 98 °C and 30 s at 58 °C. Fluorescent signals were investigated at the end of each extension step at 80 °C according to the method described by Zhang et al. [10 (link)].

For RNA extraction, plant samples were collected as follows: seedling samples were harvested at the stage when the cotyledons expanded fully. At the unifoliolate stage (when unifoliolates expand fully), hypocotyl, epicotyl, cotyledon and shoot apex (including the apical meristem and leaf primordia) materials were collected. At the flowering stage (when flowers start to open), the flowers, the 1^st, 2^nd, 3^rd and 4^th trifoliolates (from bottom to top) were harvested. The root and unifoliolate samples were collected separately at both the unifoliotate and flowering stages. The pods were sampled separately at 7, 14 and 21 days after flowering. All samples were frozen in liquid nitrogen and stored at −80°C until use.
Total RNA was extracted from seedlings, roots, hypocotyls, epicotyls, leaves, flowers, and pods using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized by using Prime Script™ RT regent Kit with gDNA Eraser (Takara, Japan). The real-time quantitative PCR was performed on a C1000 Touch TM Thermal cycler with SYBR Premix Dimer Eraser™ (Takara, Japan). Each assay was quantified in triplicate and normalized using the actin-encoding gene, glyma02g10170, as an internal control. All experiments had three biological replicates. The primers were listed in Additional file 2 (the same for other primers described below).