Quantitative RT-PCR Analysis of Arabidopsis Genes

Total RNA of various Arabidopsis tissues were extracted using Trizol reagent (Sigma) and then reverse-transcribed into cDNA with a Reverse Transcription System (TOYOBO). The cDNAs of rosette leaves of the cpnb1-4 homozygous mutants were used as the templates for PCR analysis with the gene-specific primers. qRT-PCR of CPNA2 was performed using TransStart Top Green qPCR SuperMix (TransGen, China) with a Rotor-Gene 6000 machine (Corbett Research) and the relative expression levels normalized to GAPDH were analyzed by the double standard curves method as described previously [56 (link)]. qRT-PCR of KASI was performed using TransStart Top Green qPCR SuperMix (TransGen, China) with a Bio-rad CFX Connect machine (BIO-RAD) and the relative expression levels normalized to GAPDH were analyzed by the comparative C_T method as described previously [57 (link),58 (link)]. Three biological and three technical replicates of each sample were made for qRT-PCR analysis. Primers used in the experiments were listed in S4 Table.