Quantitative Analysis of miRNA and Targets

The expression levels of randomly selected miRNAs and their targets were examined by quantitative real time PCR (qRT-PCR). Total RNA were extracted from the four different groups of samples, with three biological replications for each. The primers for the miRNAs and target genes were designed based on the methods described elsewhere [14 (link), 36 (link)]. All the sequences of primers used for qRT-PCR in this study are listed in S1 Table. The qRT-PCR analysis was subjected to CFX96 real time PCR platform (Bio-Rad, Hercules, CA, USA). The PCR conditions were 50°C for 3 min, 95°C for 5 min, then 40 cycles of 95°C for 15 sec, 55°C for 30 sec, and 40°C for 10 min. All reactions were run in triplicate. The U6 and the 18S rRNA of Paulownia were chosen as the endogenous reference genes for miRNA and target mRNA normalization, respectively. The relative expression levels of the miRNAs and targets were calculated using the method of Livak and Schmittgen [37 (link)].