Quantifying Cotton Gene Expression

Total RNA was isolated from cotton plants that were infected with Vd991 or exposed to 100 μM MeJA using an RNA extraction kit (Biomed, Enzo Life Sciences, Exeter, United Kingdom). The first strand of cDNA was synthesized from 2 μg of total RNA with a FASTQuant cDNA RT Kit (TIANGEN Biotech Co., Ltd) and used as template in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The cotton GhUBQ gene was used as an internal control. Primers were designed as shown in Supplementary Table S1. Reactions were prepared in 20-μL tubes using SYBR^®-Premix Ex Taq (Tli RNaseH Plus; Takara, Shiga, Japan) and amplified on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, United States). Expression was determined by the 2^-ΔΔCT method, and data were analyzed in Origin 8 (Liu et al., 2017 (link)).