Ssoadv univer sybr grn master kit

Total RNA was extracted from watermelon fruit tissue in three biological replicates using RNeasy^® Plant Mini Kit (QIAGEN Sciences, Germantown, MD, USA) as per the manufacturer’s protocol. The first strand cDNAs were synthesized with 1 μg of total RNA in 20 μL of the reaction mixture using iScript RT Supermix (Bio-Rad Laboratories, Inc, Hercules, CA, USA) as per the manufacturer’s instructions. Gene expression analysis via reverse transcription-qPCR was performed using the BioRad CFX96 qPCR instrument and the SsoAdv Univer SYBR GRN Master Kit (Bio-Rad Laboratories, Inc, Hercules, CA, USA). The watermelon β-actin was used as an internal control, and the relative expression levels (Cq values) for each gene were normalized to β-actin transcription. The relative expression levels were calculated using the ΔΔCq (quantitative cycle) method provided with the Bio-Rad CFX software. The primers used for qRT-PCR analysis are listed in Table S1.

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.

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.