Iscript synthesis kit

Candidate genes concerning NCLB were further screened from each QTL interval using GO and KEGG enrichment information. Candidate genes with GO terms associated with disease resistance were subjected to qRT-PCR to check their expression pattern in contrasting genotypes (resistant and susceptible). Total RNA was extracted from fresh leaves and roots using TRIzol^® Plus RNA Purification Kit (Invitrogen, CA) based on the manufacturer’s instructions. Approximately 1 μg RNA was reversely synthesized into cDNA using the iScriptTM Synthesis Kit (Quanta BioSciences, MD). The qRT-PCR was carried out in an Eppendorf real-time PCR equipment using a 5 μl cDNA template (diluted 1/100), 5 μl primers (2.4 M), and 10 μl SYBR green mixture (Promega, Madison, WI). Histone 3 was used as the internal control, and the relative expression levels of the ORP gene were calculated by the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from fresh leaves and roots using TRIzol® Plus RNA Purification Kit (Invitrogen, CA) based on the manufacturer’s instructions. Approximately 1 µg RNA was reversely synthesized into cDNA using the iScriptTM Synthesis Kit (Quanta BioSciences, MD). The qRT-PCR was carried out in an Eppendorf real-time PCR equipment using a 5 µl cDNA template (diluted 1/100), 5 µl primers (2.4 M), and 10 µl SYBR green mixture (Promega, Madison, WI). Histone 3 was used as the internal control, and the relative expression levels of the ORP gene were calculated by the 2^−ΔΔCt method [35 (link)].