4.5. Quantitative PCR/Expression Analysis of Lignin Biosynthesis Genes

Unigenes obtained from data analysis were further validated by qRT-PCR. Eight unigenes involved in lignin biosynthesis were selected for quantitative real-time expression. To evaluate gene expression, RNA was isolated from apical stem and leaves collected from plant tissue at 30 and 90 DAS. Total RNA was isolated using the Trizol reagent with 2-mercaptoethanol. RNA was further reverse transcribed, and first-strand cDNA synthesis was performed on 1 µg of total RNA using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA). Transcript analysis was performed through real-time qPCR in the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using SYBR Green SuperMix Kit (Bio-Rad, Hercules, CA, USA). Amplification was carried out through an initial step of 50 °C for 2 min, a denaturation step at 94 °C for 10 min, and 40 cycles of denaturation at 94 °C for 10 s, and annealing and extension at 60 °C for 15 s and 72 °C for 30 s, respectively. Primer pairs (Supplementary Table S1) were designed by using the Primer3 software (http://frodo.wi.mit.edu/primer3/). Expression levels were analyzed with the Eco software (ver. 3.0.16.0) and normalized versus kenaf ACT7. Relative values of expression were determined against the maximum value of individual samples at different stages. The reaction was performed in three replicates for each set of conditions and the data presented as means ± SDs (n = 3).