Fastpure plant total rna isolation kit

FastPure Plant Total RNA Isolation Kit (RC401, Vazyme) was used to extract RNA, and then we used 1µg to synthesize cDNA (HiScript III 1st Strand cDNA Synthesis Kit, R312 Vazyme). ChamQ Universal SYBR qRT-PCR Master Mix (Q711, Vazyme) was used for qRT-PCR in ABI 7500 Fast Real-time PCR System (Applied Biosystems, USA). Gene-specific primers for qRT-PCR were designed by using primer-blast in NCBI, with melting temperatures of 55–60 °C, product lengths of 101–221 bp, primer length of 18–25 bp (Table S1). For qRT-PCR, the reaction contains 10 µL 2x ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of each primer, 3 µL template, and ddH₂O to make up the total 20 µL volume. Then it was carried out in the following condition: one cycles of 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Each experiment was repeated three times, and two of the completed data were selected for drawing. Expression of all genes were calculated using a 2^−ΔΔCt method (Livak & Schmittgen, 2001 (link)).

Fusarium graminearum cultures were collected after 4–5 days of inoculation. The grains and leaves of each treatment were collected after F. graminearum was inoculated for 14 to 21 days. The extraction and purification of total RNA from plant tissues was performed with a FastPure Plant Total RNA Isolation Kit (Vazyme, Shanghai, China). Synthesis of single-stranded cDNA RT-qPCR used the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China). The RT-qPCR reaction was carried out with a ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, China) in a Roche lightcycle95 (Basel, Switzerland). The target genes of the wheat to be detected corresponded to: the disease-related proteins PR1, PR3, PR4, and PR5; the ethylene synthesis-related gene ACS; and the antioxidant enzyme gene SOD. The reference gene was β-actin. The specific primers are shown in Table 1, which was completed by Qingke Biotechnology (Beijing, China). Each treatment was replicated three times.

Total RNA was extracted using a FastPure Plant Total RNA Isolation kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. After passing the library inspection, high-throughput sequencing was then performed using the HiSeq 2000 sequencing platform at Genepioneer Biotech (Nanjing, China) according to the method of Guan et al. [57 (link)]. 104.02 Gb clean data were obtained after raw data were filtered, and the information of sequencing data was shown Table S1. The sequenced reads were assembled with Trinity software to obtain 116,944 transcripts and 58,583 unigenes. The transcript sequences were compared with genome sequence of M. cordata to assemble the cds unigenes (www.ncbi.nlm.nih.gov/nuccore/MVGT01004176), and annotations of unigenes were obtained using the NCBI (nr), Swiss-port, GO, COG, KOG and KEGG databases [9 (link), 58 (link)]. The Benjamini-Hochberg correction method is adopted to adjust the significance p value (padj), to control the proportion of false positives in the final analysis results. A padj < 0.05 and |log2 (fold change) |≥ 1 were set as the thresholds for significant differential expression between Pb treatments and the control.

Total plant RNA was isolated using the FastPure Plant Total RNA isolation Kit (Vazyme, Nanjing, China), followed by cDNA synthesis using the HiScript^® Ⅱ Q RT SuperMix for qPCR Kit (Vazyme, Nanjing, China). Primers were designed using the Primer5.0 software (Primer Premier, Oakville, ON, Canada), and their specificity was detected using the Primer-BLAST tool on NCBI. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and their sequences are outlined in Table 2. The gene expression level per treatment was the average of three biological replicates.

Total RNA was extracted from cabbage leaves at the four-leaf stage using the FastPure Plant Total RNA Isolation Kit (Nanjing, China, Vazyme) according to the manufacturer’s instructions. Genome DNA digestion and reverse transcription of the extracted RNA were carried out using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing, China, Vazyme) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was carried out with the HiScript II One Step qRT-PCR SYBR Green Kit (Nanjing, China, Vazyme) using the StepOne Plus Real-Time PCR System (Applied Biosystems). PCR primers listed in Table S7 were designed by Primer Express 3.0. PCR reaction was performed in a volume of 20 μL with the following program: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The 2^−∆∆Ct method was used to calculate relative gene expression levels [92 (link)]. BoTUB6 was used as a reference gene [93 (link)]. Three independent biological replicates with three technical repeats each were conducted.

Total RNA was extracted from cultured cells, hairy roots, and different tissues of L. erythrorhizon using the FastPure Plant Total RNA Isolation Kit (Vazyme, #RC401, Nanjing, China). The RNA purity and integrity were assessed based on the A260/A280 absorbance ratio and 1.0% agarose gel electrophoresis. cDNA was synthesized by reverse transcription with the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, #R312), and qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q711) with gene-specific primers (Table S9) on an Applied Biosystems 7500 Real-Time PCR System and StepOnePlus™ Real-Time PCR System. Gene expression levels of each sample were normalized relative to GAPDH mRNA as an internal standard and calculated using the 2^−ΔΔCt method [71 (link)]. At least three independent experiments were performed for each analysis.