Hieff qpcr sybr green master mix

RNA was extracted from the leaves of diseased plants 14 days after TuMV infection. Meanwhile, a Pure Plant Total RNA Isolation Kit was provided by TIANGEN Biochemical Technology (Beijing, China). The cDNA was then obtained in two steps using the Evo M-MLV RT Kit with gDNA Clean for qPCR (AG, Hunan, China). Then qRT-PCR was performed using 2 × Hieff^® qPCR SYBR Green Master Mix provided by YEASEN Biotechnology (Shanghai, China) to identify the relative expression levels of the genes. BcActin was used as a qRT-PCR internal reference gene. The qRT-PCR tests were used to determine the relative expression levels of genes or viruses. The primers used in this study are listed in Table S3. The data were calculated using the 2^{−∆∆ CT} method [35 (link)].

Total RNA of ‘Zhongshuang 11’ was extracted using a TransZol Up Plus RNA Kit (Transgene, Shenzhen, China) and detected using a NanoDrop™ One (Thermo Fisher, Waltham, MA, USA). cDNA was synthesized using Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China). The expression patterns of BnaGPAT9-A1/C1 in roots, stems, leaves, and developing seeds (15, 20, 25, 30, 35, and 40 days after flowering) were determined by CFX Connect™ Optics Module (Bio-Rad, Hercules, CA, USA) with three biological replications. The 20-µl qRT‒PCR contained 10 µl of 2 x Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), 0.004 nM forward primer, 0.004 nM reverse primer, and 9.2 µl of cDNA. The reaction program was initiated by predenaturation at 95°C for 5 min, and this step was followed by 40 cycles of denaturation (95°C for 10 s) and annealing (55°C for 30 s). The reference gene was ubiquitin-conjugating enzyme 9 (accession no. XM_013800933) [9 (link)], which was used to normalize the expression levels of BnaGPAT9-A1/C1. To distinguish BnGPAT9-A1/C1 by qPCR, primers were designed for the 3’ UTR and 5’ UTR differential regions of the BnaGPAT9-A1 and BnaGPAT9-C1 mRNA sequences, respectively. All primers are listed in Supplementary Table S4.

RNA extraction, reverse transcription, and PCR steps were completed on the basis of [5 (link)]. Gene-specific primers were prepared by Talen (Wuxi, China) (Table 1) [18 (link)]. Amplification of PCR was in a final reaction volume of 20 μL, including Forward Primer (0.4 μL), Reverse Primer (0.4 μL), cDNA (1 μL), RNase-Free water (8.2 μL), and 2X Hieff^® qPCR SYBR Green Master Mix (10 μL) (Cat NO.11203, Yeasen, Shanghai, China). The CT value between different storage time groups and the 0 h group were determined. The 2^−ΔΔCT method was used to calculate the gene expressions.

For RT-PCR, total RNA was isolated by TRIzol reagent (Thermo Fisher Scientific). RNA was treated with DNase and cDNA was synthesized by M-MLV Reverse Transcriptase (Promega). For real-time PCR, the reaction was performed with 2 x Hieff qPCR SYBR Green Master Mix (Yeasen) and detected by LightCycle 480 Real-Time PCR machine (Roche). For RNA-seq, RNA sample library preparation was performed according to TruSeq mRNA-seq Stranded v2 Kit (Illumina) manual instruction. The library was sequenced using Illumina Hiseq platform. Reads were aligned to GRCm38 mouse reference. HTSeq was used to quantify the aligned reads [61 (link)]. Reads normalization and differential gene expression analysis were performed using DESeq2 [62 ]. Normalized reads were subjected to Gene Set Enrichment Analysis (GSEA) [63 (link)]. For nanopore sequencing, RNA library was constructed following manual instruction (Oxford Nanopore Technologies) and the sequencing was conducted using Oxford Nanopore Technology’s MinION. Long reads were aligned to GRCm38 mouse reference using minimap2 [64 (link)]. Isoform definition and quantification were achieved by FLAIR [65 ]. Reads were visualized by Integrative Genomics Viewer (IGV) [66 (link)].

Total RNA of tissues and cells were extracted by TRIzol reagent (Invitrogen, USA). The concentration and pureness of total RNA were determined by Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen, China) was used to reverse RNA into cDNA. Hieff® qPCR SYBR® Green Master Mix (Yeasen, China) was used for qRT-PCR. 18 S, U6 and GAPDH were employed as the internal control for circRNA, miRNA and mRNA, respectively. Data were quantified by the 2^-ΔΔCt method. Primers used in this study were shown in Table S1.

Total RNA was extracted and reverse transcribed using TRIzol reagent and cDNA Synthesis SuperMix (Yeasen Biotech, China). Real-time polymerase chain reaction was carried out in Applied Biosystems QuantStudio 3 with Hieff qPCR SYBR Green master mix (Yeasen Biotech, China) and gene-specific primers.