Sybr premix ex taq 2 quantitative pcr system

To quantitatively determine the reliability of transcriptome data, the expressions of sixteen randomly selected DETs were analyzed using the qRT-PCR method. A portion of the pooled total RNA used for the RNA-Seq analysis was used to make cDNA for the qRT-PCR. The qRT-PCR was performed according to the SYBR Premix Ex Taq™ II quantitative PCR system (Takara, Dalian), following the manufacturer’s instructions, and reactions occurred on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad, Hercules, CA, USA). Gene-specific primers were designed using Primer Express software (Applied Biosystems) and are shown in Additional file 1: Table S1. Expression levels of these DETs were calculated relative to reference gene GAPDH using the 2^-ΔΔCt method [30 (link)]. Each qRT-PCR analysis was performed in triplicate, and the experiments were performed on three biological replicates.

A portion of total RNA used for the RNA-Seq analysis was used to make cDNA for qRT-PCR. qRT-PCR was conducted using the SYBR Premix Ex Taq™ II quantitative PCR system (Takara, Dalian), following the manufacturer’s instructions, and reactions occurred on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad, Hercules, CA, USA). Based on the transcriptome results, ten candidate genes involved in seed shattering were selected for the qRT-PCR assays. Gene-specific primers were designed using Primer Express software (Applied Biosystems) and are shown in Additional file 1: Table S1. Expression levels of these DETs were calculated relative to reference gene GAPDH using the 2^-ΔΔCt method [31 ]. All of the samples were tested in triplicate, and the experiments were performed on three biological replicates.