Rt reagent kit

Quantitative real-time PCR (qRT-PCR) was conducted using six genes, namely, XTH9, PER1, RAP2-3, metallothionein (MT1), RbohD, and ADH. Respective qRT-PCR primers are listed in Supplementary Table S6. Total RNA was extracted from upland cotton fiber using TRIzol Reagent. cDNA synthesis was performed using an RT reagent kit (Tiangen, China). qRT-PCR was analyzed in a 20-μl reaction system (including 10 μl SYBR Premix Ex Taq™ II (2×), 2 μl cDNA, and 0.8 μl upstream and downstream primers) and a simple procedure (50°C for 2 min; 40 cycles at 95°C for 30 s, 95°C for 5 s, and 60°C for 20 s; and a final extension at 72°C for 10 min). Three biological repeats were included for each condition, and the GhUBQ gene was used as a control. The relative expression levels were calculated using the 2^-ΔΔCt method.

The qRT-PCR primers are listed in S1 Table. Total RNA was extracted from upland cotton leaves at different developmental stages using TRIzol Reagent. cDNA synthesis was performed using an RT reagent kit (Tiangen, China). qRT- PCR was analyzed in a 20-μL reaction system (including 10 μL SYBR ® Premix Ex Taq ™ Ⅱ (2 x), 2 μL cDNA, and 0.8 μL upstream and downstream primers) and a simple procedure (50°C for 2 min; 40 cycles at 95°C for 30 s, 95°C for 5 s, and 60°C for 20 s; and a final extension at 72°C for 10 min). The GhUBQ gene was used as a control, and each sample was repeated 3 times. The relative expression levels were calculated using the 2^−Ct method. All experiments included three biological replicates.

Total RNA was extracted using Trizol (Vazyme). For the mRNA analysis, the cDNA primed by oligo-dT was made with RT reagent kit (Tiangen, China), and the mRNA level of Rab27B was quantified by a duplex-qRT-PCR analysis where the TaqMan probes with a different fluorescence for β-actin (Shing Gene, China) were used in the FTC-3000P PCR instrument (Funglyn, Canada). The miRNA expression level was normalized using U6 small nuclear RNA (HmiRQP9001) as an internal control, as previ-ously described [22 (link)]. Using the 2^−ΔΔCt method, the β-actin level was normalized before comparing the relative level of the target genes. The sequences of primers and probes used for the qRT-PCR analysis are as follows:
hRab27BF, 5′-GGGACACTGCGGGACAAG-3′;
hRab27BR, 5′-CAGTTGGCTCATCCAGTTTCTG-3′;
hRab27B probe, 5′-ROX-CGGTTCCGGAGTCTCACCACTGC-3′;
hACTB F: 5′-GCCCATCTACGAGGGGTATG-3′
hACTB R: 5′-GAGGTAGTCAGTCAGGTCCCG-3′
hACTB probe: 5′-CY5-CCCCCATGCCATCCTGCGTC-3′