Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assays were performed to validate the accuracy of transcriptome sequencing data. After removing genomic DNA from RNA samples with RNase-free DNase I, total RNA was used as RT templates for cDNA synthesis using the PrimeScript™ RT Reagent Kit Eraser (Perfect Real Time; TaKaRa, Shiga, Japan). The RT-qPCR assays were performed to detect relative gene expression using SYBR®Premix Ex Taq™ II (TliRNaseH Plus) (TaKaRa, Shiga, Japan) using a Bio-Rad C1000 touch Thermal Cycler of CFX96™ Real-time PCR detection System.
The RT-qPCR program was as follows: 95 °C for 3 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The melting curve was plotted as follows to analyze the primer gene-specificity: 95 °C for 15 s, 60 °C for 1 min, and 60–95 °C for 15 s (+ 0.3 °C/cycle). The expression data of target genes were normalized using two public internal reference genes, BnaEF1-α and BnaGDI1 [70 (link)], and the relative gene expression levels were calculated according to the 2-ΔΔCT method [71 (link)]. Each sample contained three independent biological replicates.
The RT-qPCR program was as follows: 95 °C for 3 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The melting curve was plotted as follows to analyze the primer gene-specificity: 95 °C for 15 s, 60 °C for 1 min, and 60–95 °C for 15 s (+ 0.3 °C/cycle). The expression data of target genes were normalized using two public internal reference genes, BnaEF1-α and BnaGDI1 [70 (link)], and the relative gene expression levels were calculated according to the 2-ΔΔCT method [71 (link)]. Each sample contained three independent biological replicates.