Sybr premix ex taqtm kit

To verify the accuracy and repeatability of the RNA-seq data, 15 DEGs were selected for qRT-PCR validation. The total RNA was extracted from the first true leaf on the 14th d after Pi deficiency treatment by using RNAprep Pure Plant Plus Kit (Tiangen Biotech, Beijing, China), and cDNA was synthesized using PrimeScript^TM RT Master Mix. Quantitative RT-PCR was performed using an SYBR^® Premix Ex TaqTM Kit (Tiangen Biotech, Beijing, China) with a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) with six replicates. The relative expression was calculated using the 2^−∆∆Ct method [58 (link)]. Actin was used as an internal control [59 (link)]. The primers used for qRT-PCR were listed in Supplementary Table S1.

Total RNA was isolated respectively from frozen liver tissues and cells by TRIzol reagent (Invitrogen, USA), and then up to 1 µg of RNA was reverse transcribed by Prime Script RT Master Mix Kit (Tiangen Biotech, Beijing, CN). cDNA was duplicated by SYBR Premix ExTaqTM Kit (Tiangen Biotech, Beijing, CN). The primer sequences are listed in Supplementary Table 1. Data were analyzed using the 2^−ΔΔCt method and normalized to β-actin expression as previously described.