Validating RNA-seq Data via RT-qPCR

To validate the RNA-seq data, quantitative real-time PCR (RT-qPCR) was conducted to examine the expression pattern of twenty DEGs. Primer Premier 3.0 software was used to design gene-specific primers on the basis of the selected unigene sequences (S1 Table). Total RNA was extracted with the Quick-RNA^™ Miniprep Kit (Zymo Research Corporation, Irvine, CA) treated with DNase1 (Zymo Research Corporation, Irvine, CA), and subjected to reverse transcription using iScript RT Supermix (Bio-Rad Laboratories, Inc, Hercules, USA). The quality and quantity of the RNA were analyzed by a Denovix DS-11+ spectrophotometer (Wilmington, Delaware, USA). Gene expression analysis via reverse transcription-qPCR was performed using the BioRad CFX96 qPCR instrument using SsoAdv Univer SYBR GRN Master Kit (Bio-Rad Laboratories, Inc, Hercules, USA). The expression levels of selected DEGs were normalized by comparing with an internal reference gene, 18SrRNA [40 (link)]. The relative expression levels (Cq values) for each gene were normalized to that of reference genes by taking an average of three biological replicates. The relative expression levels were calculated using the 2^−ΔΔCt method [41 (link)]. All RT-qPCR were repeated in three biological and three technical replications.