Validating RNA-seq Data via RT-qPCR

To validate the RNA-seq data, total RNA was extracted from three replicate leaf tissues of salt-stressed seedlings (Crimson Sweet). The expression pattern of selected DEGs was examined using quantitative real-time PCR (RT-qPCR). The gene-specific primers based on the selected unigene sequences (Table S9) were designed using Primer Premier 3.0 software. Total RNA was extracted with the Quick-RNA™ Miniprep Kit (Zymo Research Corporation, Irvine, CA, USA) followed by DNase1 (Zymo Research Corporation, Irvine, CA, USA) treatment, and subjected to reverse transcription using iScript RT Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA ). The quality and quantity of the RNA were examined using a Denovix DS-11+ spectrophotometer (DeNovix Inc. Wilmington, DE, USA). Gene expression analysis via reverse transcription-qPCR was performed using a BioRad CFX96 qPCR instrument and by using a SsoAdv Univer SYBR GRN Master Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Watermelon β-actin and α-tubulin5 genes [123 (link)] were used as the internal controls, and the relative expression levels (Cq values) for each gene were normalized by taking an average of three biological replicates. The relative expression levels of each gene were calculated using the 2^−ΔΔCt method. The primers for qPCR used in this chapter are listed in Supplementary Materials, Table S9.