Total RNA was isolated with an RNAprep Pure Plant Plus kit (Tiangen Biotech, China) and used as the template for reverse transcription with a PrimeScript RT reagent kit (TaKaRa, Japan). The RT-qPCR assays were performed using the UltraSYBR Mixture (Cwbio, China). The cotton GhSSU (small subunit ribosomal rRNA) gene, the Arabidopsis AtUBC21 (Ubiquitin-Conjugating Enzyme 21) gene, and the N. benthamiana and V. dahliae EF-1α (Elongation Factor-1α) genes were used as internal controls. The setting of thermal cycler was: predenaturation at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. A final reaction at 72°C for 10 min was applied. Melt curves were analyzed to ensure there was not nonspecific amplification. Relative transcript levels among various samples were determined using the 2–ΔΔCT method, with three independent determinations (58 (link)). The sequences of primers used in this study are listed in Table S4.
Protocol detail
Quantifying Relative Gene Expression Using RT-qPCR
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Arabidopsis
Assays
Cotton
Elongation factor 1α
Genes
Plant
Primers
Reverse transcription
Ribosomal
Rrna gene
Subunit
Ubiquitin conjugating enzyme
Corresponding Organization : Northwest A&F University
Other organizations : Chinese Academy of Agricultural Sciences, Institute of Plant Protection, Agricultural Research Service, United States Department of Agriculture, University of California, Davis
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Variable analysis
independent variables
- None explicitly mentioned
- Relative transcript levels among various samples
- Total RNA was isolated with an RNAprep Pure Plant Plus kit
- Reverse transcription was performed with a PrimeScript RT reagent kit
- RT-qPCR assays were performed using the UltraSYBR Mixture
- The cotton GhSSU (small subunit ribosomal rRNA) gene, the Arabidopsis AtUBC21 (Ubiquitin-Conjugating Enzyme 21) gene, and the N. benthamiana and V. dahliae EF-1α (Elongation Factor-1α) genes were used as internal controls
- Thermal cycler settings: predenaturation at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, with a final reaction at 72°C for 10 min
- Melt curves were analyzed to ensure there was not nonspecific amplification
- None explicitly mentioned
- None explicitly mentioned
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