Quick rna isolation kit

Quick RNA isolation Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) was used to extract RNA according to the manufacturer’s instructions and DNase I treatment was used to remove DNA contamination. Synthesis of the first strand of cDNA was carried out according to the instructions of the kit (TaKaRa, Beijing, China). mRNA and lncRNA were detected by TB Green Premix Ex Taq II (TliRNaseH Plus) and ROX plus (TaKaRa, Beijing, China). The first-strand cDNA synthesis reagent of miRNA was performed simultaneously by A addition method and reverse transcription reaction, and NovoScript^® miRNA First-Strand cDNA Synthesis and SYBR qPCR Kit (Suzhou Nearshore Protein Technology Co., Ltd., Suzhou, China) was used. qRT-PCR reactions were conducted using the following protocol: 95 ˚C for 2 min, followed by 40 cycles of 95 ˚C for 20 s and 60 ˚C for 20 s and 72 ˚C for 20 s. The wheat elongation factor (TaEF-1a) gene was used as an internal reference for mRNAs and lncRNAs expression analyses. miRNAs expression analyses using TaU6 gene as an internal reference gene. Threshold values (CT) were generated using the ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA), and the transcript levels assessed using the comparative 2^-ΔΔCT method. All primers used in this study are listed in supplementary Table S1.

The total RNA concentration of the apple pulp samples were extracted through a Quick RNA isolation kit (Tiangen, Beijing, China), and the cDNA reverse transcription was performed using the All-in-One First-Strand cDNA Synthesis SuperMix with gDNA Eraser (TransGen Biotech, Beijing, China). Using the ABI7500 System and SYBR Green Master Mix (Vazyme, Nanjing, China), a RT-qPCR was performed. Actin served as an internal control. The primers for the RT-qPCR are shown in Table S1. The genebank accession numbers are shown in Table S2.

The Quick RNA isolation Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) was used to extract RNA according to the manufacturer’s instructions, and DNase I treatment was used to remove DNA contamination. Synthesis of the first strand of cDNA was carried out according to the instructions of the kit (Takara, Japan). To measure the expression of TaELPs, gene-specific primers were designed using WheatOmics 1.0 PrimersServer Database (http://202.194.139.32/PrimerServer/, accessed on 21 October 2021). qRT-PCR analysis was performed with specific primers (Table S10). qRT-PCR reactions were conducted using the following protocol: 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 20 s and 72 °C for 20 s. We verified the specificity of each primer’s amplicon via melting curve analysis, and the Elongation factor 1a (TaEF-1a) was used as an internal reference gene (GenBank accession no. Q03033) [85 (link)]. The threshold values (CT) were generated using the ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA), and the transcription level of TaELPs was assessed using the comparative 2^−ΔΔCT method [86 (link)].