Plant total rna extraction kit

Transgenic and WT plants grown in soil pots for one month were treated with 150 mM NaCl for 24 h. Three overexpression transgenic lines (OC), three RNA interference transgenic lines (RC), and one wild-type control top leaf (WT) were collected, immediately frozen in liquid nitrogen, and saved in a −80 °C freezer for similar evaluation and sequencing (each strain had three biological replicates). Total RNA was extracted using the plant total RNA extraction kit (TIANGEN, Beijing, China). The purity and integrity of RNA were checked by nanodrops, agarose gel electrophoresis and the Agilent Biological Analyzer 2100 system (Agilent Technologies Co. Ltd., Beijing, China).Total RNA was extracted and sent to Gene Denovo (Guangzhou, China) for sequencing using the Illumina HiSeq2000 platform. The experimental procedure for transcriptome sequencing included library construction, library quality control and up-sequencing.

The transcriptome data of GbCDPK genes in the tissues and the abiotic stresses were from Sequence read archive, SRA, and the accession number was PRJNA274882. The RPKM value (the reads per kilobase of transcript per million mapped reads) represents the abundance of gene expression at the transcription level. Using Tbtools (Chen et al., 2020 (link)) map the gene expression profiles of different tissues and different abiotic stresses. Total RNA is extracted with the plant total RNA extraction kit (TIANGEN, Beijing, China). RNA reverse transcription adopts HiScript II QRT SuperMix for qPCR (R222-01), a product of Nanjing Novazin. The cDNA was used as a template to perform RT-PCR and qPCR experiments. The internal reference gene is the ubiquitinated protein Ubiquitin7 (UBQ7), and the RT-PCR reaction uses 1.0% agarose gel to detect the amplification results. The quantitative instrument uses Roche products, and the calculated Ct value is used to calculate the relative expression of the gene. The calculation method is 2^−ΔΔCt (Pfaffl, 2001 (link)). Each gene amplification result contains at least 3 technical replicates and 3 biological replicates. The amplification system and procedure are shown in Appendix A and Appendix B.

Total RNA was isolated from the collected plant tissues using a plant total RNA extraction kit (Tiangen, China, Cat DP432) in accordance with the manufacturer’s instructions, and treated with DNase I (Tiangen, China, Cat DP432). The quality and quantity of RNA were measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from the total RNA using a cDNA Synthesis Kit (Tiangen, China, Cat KR106) as per the manual. The PagSTOMAGEN coding sequence was cloned using the PrimeStar^® High-fidelity Thermostable DNA Polymerase Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, Liaoning, China), and the primers are shown in Supplementary Table S1.

Oligo7 was used to design RT-PCR primers for TaPLC genes. Total RNA was extracted using the plant total RNA extraction kit (TIANGEN, Beijing, China). The cDNA was synthesized with a First Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). 18S RNA was used as the internal reference gene [28 (link)], and the specific primer sequences of each gene are shown in Table S4. Reactions of 20 µL contained 2× Super-Real Mix 10 µL, 50× ROXII; 2 µL, forward and reverse primers 1 µl for each, cDNA 2µL, and ddH₂O 4 µL. Reaction conditions included pre-denaturation at 95 °C for 15 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 32 s, and 40 cycles. Three biological replicates and three technical replicates were applied for all qPCR analyses in this study. The 2^-ΔΔCT method was used to calculate relative gene expression. Software SPSS 19 was used for significance analysis and standard deviation calculation.

To confirm the candidate gene expression pattern, a plant total RNA extraction kit (Tiangen, Beijing, China) was used to extract total RNA from roots and leaves at the fifth true leaf stage of ‘FT’ and cwm seedlings. The FastQuant RT Super Mix (Tiangen, Beijing, China) was used to synthesize cDNA. The primer RT-1 designed by Primer Premier 5.0 (Table S3) and the SYBR Green PCR Master Mix (Takara Bio Inc., Kusatsu, Japan) were used for qRT-PCR. The data were analyzed using QuantStudio^TM Real-Time PCR software. The program and reaction volume of the qRT-PCR were described previously [42 (link)].

Total RNA samples were extracted from fresh leaves of different stages (cotyledon, seedling, rosette, and heading stages) using a plant total RNA extraction kit (Tiangen, Beijing, China). cDNA was synthesized using FastQuant RT Super Mix 13 (Tiangen, Beijing, China) Quantitative real-time PCR (qRT-PCR) amplification was carried out in QuantStudio 6 (Life Technologies, California, USA) using SYBR Green PCR Master Mix (Takara Bio Inc., Kusatsu, Japan) in a 20 μl reaction mixture. Gene-specific primers were designed using Primer Premier 5.0, and the ACTIN gene was used as the internal control (Supplementary Table S8). The qRT-PCR amplification reaction system and procedure was described as Huang et al.^{48 (link)}.

Two to three leaves were taken from each variety and the total RNA was extracted using the plant total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China), and the genomic DNA in the total RNA was removed by DNase I (Tiangen Biochemical Technology Co., Ltd., Beijing, China). A total of 1 μg of RNA was used as template. The first strand of cDNA was synthesized using the Prime Script RT reagent Kit (Ta Ka Ra, Dalian, China).
The con-infection of SPFMV and SPCSV was detected by RT-qPCR method, using the primers developed by Lu et al. (Table 4) [27 ]. RT-qPCR was performed using SsoAdvanced PreAmp Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a Bio-Rad CFX96 Touch PCR Detection System with the following conditions: 95 °C for 10 min and then 45 cycles of 95 °C for 15 s and 58–66 °C for 30 s, followed by a melt cycle of 65 °C for 5 s and 95 °C for 15 s [39 (link),40 (link)]. Reactions were performed in triplicate, with a negative nuclease-free water control in each run. Sweet potato H2 B and UBI encoding genes were used as a double internal control for normalization of the gene expression data [41 (link)]. The relative expression levels of virus were quantified with the delta threshold cycle (ΔCt) method [42 (link)], referenced to the internal control. The experiments were repeated three times in independent RT-qPCR reactions.