Cfx96 real time fluorescent quantitative pcr detection system

TRIzol (Invitrogen) was used to extract RNA, and then 1μg of RNA was reversely transcribed into cDNA using the PrimeScript™ RT kit (Takara) according to the instructions. The SYBR Green kit and CFX96 real-time fluorescent quantitative PCR detection system (Bio-Rad, Richmond, CA, USA) were used for qPCR. Taking 18S as internal reference, the Ct (18S-PANK1) method was used to calculate the expression of the target gene. See Supplementary Table 2 for details on the primer sequence.

To examine the reproducibility and accuracy of DEGs based on transcriptome sequencing, six DEGs were randomly selected for qPCR validation. Total RNA was extracted from maize seed radicle samples (CT or TS) using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA). To determine RNA quality, a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used with a 1% agarose gel. Primers targeting these six genes were designed using Primer Premier 5.0 with ZmActin [44 (link)] as the internal reference gene (Table S1). A CFX96 real-time fluorescent quantitative PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used. The PCR cycling conditions included 60 s of initial denaturation at 95 °C, followed by 15 s of denaturation at 95 °C and 30 s of annealing at 55 °C for a total of 39 cycles. Three biological replicate samples were included in each treatment (each biological replicate contained three technical replicates), and gene expression levels were calculated according to the 2^−∆∆Ct method [45 (link)].