Easyspin plus rnaprep kit

Total RNAs of all the collected samples were extracted using EASYspin Plus RNAprep Kit (Aidlab, Beijing, China). The quantity and quality were determined by a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Dalian, China). All the protocols followed to the manufacturer's instructions. qRT-PCR was performed with Lightcycler 96 system (Roche, Mannheim, Germany) using SYBR the premix Ex taq (TakaRa, Dalian, China) in 20 μL volume according to the supplier's protocols. The specific primers used were listed in Supplementary Table 1, and cotton UBQ7 was used as an internal control. Three biological replicates were performed for each sample. The relative expression levels were calculated according to the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). The heatmap for expression profiles were generated with the Mev 4.0 software (Saeed et al., 2003 (link)).

Fifty-eight pairs of ALDH gene specific primers from G. hirsutum were used to study the expression profile of ALDH gene superfamily by qRT-PCR. Total RNAs of all the collected samples were isolated using EASYspin Plus RNAprep Kit (Aidlab, Beijing, China). A NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to detect the quantity and quality of total RNAs. Approximately 500 ng RNA was reverse transcribed using the PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Dalian, China) to synthesis cDNA. All the protocols were followed the manufacturer’s instructions. qPCR was performed with Lightcycler 96 system (Roche, Mannheim, Germany) using SYBR the premix Ex taq (TakaRa, Dalian, China) in 20 μL volume according to the supplier’s protocols. The specific primers used in current research are listed in S1 Table. G. hirsutum UBQ7 was used as internal control to normalize all data. Each gene was run in triplicate from three biological replicates. 2^−ΔΔCt method was carried out to calculate the relative expression levels [40 (link)]. And the heatmap for expression profiles were generated with the Mev 4.0 software [41 (link)].