Plant DNA and plasmid DNA isolation was performed using a plant DNA isolation kit (Tiangen Biotech) and a plasmid DNA extraction kit (Tiangen Biotech), respectively, following the manufacturer’s instructions. For RNA isolation, all tissues were collected fresh, flash-frozen in liquid nitrogen, and total RNA was extracted using a plant total RNA isolation kit (Tiangen Biotech) following the manufacturer’s instructions. First-strand cDNA was synthesized using a reverse transcription kit (TAKARA) as directed by the manufacturer. For qPCR, 2 μl of cDNA was used as template in 20 μl reaction volumes, and the qPCR amplifications were performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus; TAKARA) on a LightCycler® 480 instrument (Bio-Rad). The rice UBIQUITIN10 (OsUBQ10) or ACTIN1 genes were used as the internal controls for normalization of gene expression. All oligonucleotide primers used in this study are given in Supplementary Table 1 .
Plant total rna isolation kit
Manufactured by Tiangen Biotech
Sourced in China
The Tiangen Biotech Plant total RNA isolation kit is designed for the extraction and purification of total RNA from various plant tissues. The kit utilizes a guanidinium-based lysis buffer and silica-based columns to efficiently capture and purify high-quality RNA suitable for downstream applications such as RT-PCR, Northern blotting, and RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcription kit, by Takara Bio (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Plant dna isolation kit, by Tiangen Biotech (1 mentions)
Plasmid dna extraction kit, by Tiangen Biotech (1 mentions)
Lightcycler 480 instrument, by Bio-Rad (1 mentions)
Transscript all in one first strand cdna synthesis supermix for qpcr kit, by Transgene (1 mentions)
Takara sybr green mix kit, by Takara Bio (1 mentions)
Abi 7500 fast real time detection system, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Tiangen Biotech (1 mentions)
Dnbseq platform, by MGI Tech (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Hiseq platform, by Illumina (1 mentions)
5 protocols using plant total rna isolation kit
1
Plant DNA, RNA, and cDNA Isolation and qPCR Analysis
2
Quantifying Soybean SOD Gene Expression
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Total RNA was extracted from soybean using the plant total RNA isolation kit (TIANGEN, Beijing, China), and the cDNAs were synthesized using the TransScript All-in-One First-Strand cDNA synthesis SuperMix for qPCR kit (TransGen Biotech, Beijing, China). qRT-PCR assays were performed using UtraSYBR Mixture (low ROX) and ABI 7500 sequencer. The GmGADPH was used as internal control (Huis, Hawkins & Neutelings, 2010 (link)). The SOD genes and GmGADPH primers are listed in supplementary Table S1 . The three biological replicates were obtained and expression levels were calculated using 2−ΔΔCt method and Student’s t-test (Livak & Schmittgen, 2001 (link)).
3
Validating Transcriptome Findings via qRT-PCR
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Nine genes were selected from the DEGs and detected using real-time quantitative techniques to verify the correctness of the transcriptome results. The RNA of each sample was extracted using a plant total RNA isolation kit (Tiangen, Beijing, China) based on the manufacturer’s instructions. The cDNA of all samples was obtained using the Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), and the specific operation steps followed the instructions of the manufacturer. In addition, qRT–PCR was performed on the ABI 7500 Fast Real-Time Detection System by using the TaKaRa SYBR Green Mix kit (TaKaRa, Kyoto, Japan). The PCR amplification experiment was performed with 10 μL 2 × SYBR Green premix ExTaq II, 0.4 μL Rox Reference Dye II, 0.8 μL primer-F/R, 2 μL cDNA and ddH2O to 20 μL. Then, qRT–PCR was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 35 s, 95 °C for 5 s, 60 °C for 1 min and 95 °C for 15 s. The reference gene was 18S-RNA, and all primers are listed in Supplementary Table S18 . The relative expression analysis of quantitative data was performed using the 2−ΔΔCT method [95 (link)].
4
Transcriptional Profiling of Grapevine Pathogens
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The collected leaves were inoculated as described previously. We chose infected grape leaves 24 and 48 h post-infection of VvMF and Vd0940, and non-infected leaves (0 h) as a control, using three replicates per cultivar (Supplementary Data Fig. S2 ). Total RNA was extracted from entire leaves of VvMF and Vd0940 using the Plant Total RNA Isolation Kit (DP441, Tiangen Biotech, China) in accordance with the manufacturer’s instructions. Subsequently, cDNA was synthesized using the PrimeScript™ RT Reagent Kit (KR116, Tiangen Biotech, China). RNA sequencing was performed using the DNBSEQ platform (MGI Tech, China). High-quality clean reads were obtained by filtering raw reads using SOAPnuke. The clean reads were then compared with the reference gene sequence using Bowtie2. Gene expression levels were calculated using RNA-Seq by Expectation Maximization (RSEM) [46 (link)] and were expressed as FPKM. The datasets can be found at NCBI under BioSample accession PRJNA938012. To evaluate the transcription levels of VvMF and Vd0940 candidate genes, qPCR primers (qRTPCR-PR1, VvActin) were designed using Primer3plus (https://www.primer3plus.com/ ). The expression level was determined as 2−ΔΔCt, normalized to the Ct value of VvActin [47 (link)]. Details of the primers utilized are provided in Supplementary Data Table S1 .
5
Transcriptome Analysis of Plant Samples
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The total RNA from each sample was extracted using a plant total RNA isolation kit (Tiangen, Beijing, China) based on the manufacturer’s instructions. A total of nine cDNA libraries (three replicates each of CK, LS and HS) were constructed. After estimating the quality, concentration and integrity of total RNA, the RNA was sequenced on the Illumina HiSeq platform (HiSeqTM 2500, San Diego, CA, USA). To ensure the quality of the raw reads, they were filtered using Fastp [94 (link)] software (version 0.12) to obtain high-quality clean reads. De novo assembly of unigenes was performed using Trinity [29 (link)] software (v2.11.0). The longest transcript at each locus was considered a unigene, and the unigene IDs were automatically generated by the software. Gene functional annotation of the assembled unigenes was performed using public databases, including Nr [30 ], Swiss-Prot [31 (link)], GO [32 (link)], KOG [33 (link)], KEGG [34 (link)], Pfam [35 (link)] and Trembl databases.
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