Plant total rna extraction kit

For fungal growth biomass, fungal DNA was extracted using five cotton stems through the CTAB method at 3 weeks after V. dahliae infection (Muhammad et al., 2013 (link)). Total RNA from cotton samples, including the roots, internodes, leaves, cotyledons, and hypocotyls, was extracted using a plant total RNA extraction kit (Sangon, Shanghai, China) following the manufacturer’s protocol. First-strand cDNA was synthesised from 3 μg of total RNA using the Goldenstar RT6 cDNA Synthesis kit Ver. 2 (Tsingke, Beijing, China) to analyse the expression levels of related genes by qRT-PCR. Quantitative detection was performed using the 2 × T5 Fast qPCR mix kit (Tsingke, Beijing, China) in a real-time quantitative PCR instrument (Bio-Rad, Foster City, CA, United States) and the relative quantification was calculated using the comparative Ct method (Livak and Schmittgen, 2001 (link); Li et al., 2005 (link)). To normalise gene expression, GhUB-7 was used as an internal control. The experiments were performed with three technical replicate and three biological replicates (n ≥ 3). All primers used in the experiments are shown in Supplementary Table S1.

Total RNA of the cotton leaves and roots was isolated using the Plant Total RNA Extraction Kit (Sangon Biotech, Shanghai, China) according to the instruction manual. Two micrograms of RNA was reverse transcribed for first strand cDNA synthesized following the manufacturer’s protocol using the TransScript First-Strand cDNA Synthesis kit (TransGen).
According to the protocols of the Minimum Information for Publication of Quantitative Real Time PCR Experiments (Bustin et al., 2009 (link)), diluted cDNA was used for qPCR with SYBR green using the CFX96 Touch^TM Real-Time PCR detection systems (Bio-Rad, Foster City, CA, United States). All gene expression was calculated using the dCt or ddCt algorithm. To normalize the gene expression, the UB7 gene was used as an internal standard. All the gene specific primers involved in this study were listed in Supplementary Table S1.

The correlation of mRNA levels and promoter strengths selected strains harbor different promoters were determined with their corresponding mRNA levels and promoter strengths. The method for determination of the promoter strengths was as described above. The mRNA levels were determined by real-time quantitative PCR (RT-qPCR). Total RNA were extracted with plant total RNA extraction kit (Sangon, Shanghai). After reverse transcription with the reverse transcription kit (Takara Ltd, Dalian, China), the mRNA level was determined by real-time RT-PCR kit (Bio-Rad). The mRNA levels of yEGFP in different strains were determined with ALG9 as the internal control gene [47 (link)], and the primers for detection of ALG9 and yEGFP were listed in Additional file 1: Table S1.