Expression analysis of flavonoid biosynthesis gene families in safflower

In general, semi-quantitative PCR is used when the accuracy of gene expression is not high and the expression within differential treatments show a big difference. It was used more in tissue-specific expression analysis. Here, we analysed the expression of the annotated flavonoid biosynthesis genes using semi-quantitative RT-PCR. All gene specific primers were designed to amplify products of 100–400 bp in length. The length of the primers was 20 ± 2 bp. The specific primer for the 28S gene was used as the internal control. The specificity was tested by agarose gel electrophoresis. The detailed PCR primer sequences are shown in Additional file 1: Table S1. RNA was isolated from roots, stems, leaves, and three developmental stages of safflower. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions and was treated with DNase I (Takara). First-strand synthesis of cDNA was performed using RR047 kit (Takara) (1 μg of total RNA was used for cDNA synthesis in a 20 μl reaction volume). In order to adjust the PCR reaction for flavonoid biosynthesis gene, the amount of the template was adjusted, and then the semi-quantitative PCR reaction of the flavonoid biosynthesis gene was carried out. The expression of each flavonoid biosynthesis gene was detected by gel electrophoresis.