The leaves (HS-24, 48, and 72 h, HJ-24, 48, and 72 h), mycelium (JS-3 days, 6 days), and conidia were taken into a mortar, frozen in liquid nitrogen and well ground, and 10–20 μg of different sample powder were weighed and added to 1.5 ml sterile enzyme-free centrifuge tube, and different samples of RNA were extracted using RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich; TianGen, Beijing, China). Extracted RNA was determined by Eppendorf Biophotometer D30 (Eppendorf, Hamburg, Germany) for purity and concentration. Sample RNAs were required with A260/A280 ratios of 1.9 to 2.1 and A260/A230 ratios of 19 to 2.0, indicating high purity and no protein contamination of samples. RNA integrity examines by 1% agarose gel electrophoresis. Approximately 100 ng of total RNAaspirate for cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (Vazyme, Nanjing, China).
Rnaprep pure plant plus kit polysaccharides and polyphenolics rich
Manufactured by Tiangen Biotech
Sourced in China
The RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) is a laboratory equipment product designed for the efficient extraction and purification of RNA from plant tissues that are rich in polysaccharides and polyphenolics. The kit employs a specialized procedure to ensure the removal of these interfering compounds, allowing for the isolation of high-quality RNA suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiscript 3 1st strand cdna synthesis kit gdna wiper kit, by Vazyme (1 mentions)
Biophotometer d30, by Eppendorf (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (1 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hiscript 2 q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Nanophotometer spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Noveseq 6000 platform, by Illumina (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Perfectstart green qpcr supermix, by Transgene (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Cfx96 real time system thermal cycler, by Bio-Rad (1 mentions)
Easyscript one step gdna removal and cdna synthesis supermix kit, by Transgene (1 mentions)
Abi prism 7500 sequence detector, by Thermo Fisher Scientific (1 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Takara tb green premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Mircute mirna isolation kit, by Tiangen Biotech (1 mentions)
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
8 protocols using rnaprep pure plant plus kit polysaccharides and polyphenolics rich
1
RNA Extraction from Fungal Samples
2
Quantitative Gene Expression Analysis in Plants
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Total RNA was extracted using an RNAprep Pure Plant Plus Kit (polysaccharides and polyphenolics rich; TIANGEN, Beijing, China). The first cDNA strand was synthesized with 2 μg of total RNA by using a FastKing RT Kit (with gDNase) (TIANGEN, Beijing, China), as per the manufacturer’s instructions. qRT-PCR was performed with the CFX96 Touch C1000 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) by using specific primers designed by Primer 5 Software (Table S3 ). Three biological and three technical replicates for each sample were performed with 20 µL of reaction volume using the SYBR® Green Realtime PCR Master Mix (Toyobo, Osaka, Japan). The ACTIN gene was used as the housekeeping loading reference. The relative gene expression was calculated using the 2−ΔΔCt method [73 (link)].
3
Transcriptional Response of Wheat to Fusarium graminearum
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Common wheat WSB, AN1589, and AN8455 were planted in the field. Each wheat variety was injected with 15 μl of F. graminearum (mixed strain) conidia suspension or water between the lemma and palea of the wheat spikelet at anthesis. Samples were taken at 12, 24, 36, 48, and 72 h after inoculation, and frozen in liquid nitrogen and stored in the refrigerator at −80°C.
Total RNA was extracted from wheat spikelets infected by F. graminearum using RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) (TIANGEN, China) according to the manufacturer’s instruction. The total RNA was reverse transcribed into cDNA using HiScript® IIQRT SuperMix for qPCR (+gDNAwiper) (Vazyme, China). The oligonucleotide primer pairs used in the qRT-PCR analyses were listed in theSupplementary Table 12 . Actin (AB181991) was used as the internal reference gene. The qRT-PCR was carried out using AceQ® Universal SYBR® qPCRMasterMix (Vazyme, China) according to the manufacturer’s instructions. Each treatment was repeated three times. The relative expression was determined by using the 2–ΔΔCT method (Schmittgen and Livak, 2008 (link)).
Total RNA was extracted from wheat spikelets infected by F. graminearum using RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) (TIANGEN, China) according to the manufacturer’s instruction. The total RNA was reverse transcribed into cDNA using HiScript® IIQRT SuperMix for qPCR (+gDNAwiper) (Vazyme, China). The oligonucleotide primer pairs used in the qRT-PCR analyses were listed in the
4
RNA Extraction and RNA-seq Library Preparation
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Total RNA was extracted using the RNAprep Pure Plant Plus Kit (polysaccharides and polyphenolics-rich) (Tiangen, Beijing, China). The purity and concentration of RNA were measured using a NanoPhotometer spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity and quantity of RNA were assessed using an Agettent 5400 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Next, library preparation was performed. Total RNA was input, and mRNA was captured by mRNA capture beads with oligo (DT), purified in buffer to a random fragment of 100–200 nt, and reverse transcribed into cDNA subsequently. Ligating purified cDNA to premixed adaptors with UMI using ligase and ligase buffer was followed by one-step polymerase chain reaction. After purification, the PCR amplification products were recovered. The library was sequenced on the Illumina noveseq 6000 Platform to obtain 6G of raw data, yielding 150 nt of pair-end reads.
5
RNA Extraction and RT-qPCR Analysis Across Plant Species
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The total RNA of P. betulaefolia, M. domestica and tobacco was extracted with RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics rich) from Tiangen Biotech company, according to the manufacturer’s instructions. To avoid genomic DNA contamination, extracted RNA was treated with DNase I. RNA was reverse transcribed into cDNA by the Aidlab Bio’s TRUESCript 1st Strand cDNA Synthesis Kit. RT-qPCR assay of all tissues was carried out on Thermo Fisher Scientific’s stepone Plus real-time PCR instrument with Tiangen’s SuperReal premix Plus. The relative expression levels were normalized to the internal control PbActin, MdActin and NbActin in P. betulaefolia, M. domestica and N. benthamiana, respectively. All primers used are listed in Table S1 .
6
Quantifying AktDofs Gene Expression
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Total RNA was extracted with an RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) (TIANGEN, Beijing, China). The integrity and purity of the RNA were checked via an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Austin, TX, USA), respectively. Then, the RNA of the samples was reverse transcribed into cDNA using an EasyScript One-Step gDNA Removal and cDNA Synthesis Supermix Kit (TransGen Biotech, Beijing, China). The primer pairs for the AktDofs and GAPDH gene were designed using Primer 3.0, and the primer sequences and related details are listed in Table S6 . The amount of cDNA was 1μmol as the amplification substrate, and the reaction was carried out as follows: 92 °C for 30 s, followed by 45 cycles of 5s at 92 °C, and 30 s at 60 °C. To determine the expression patterns of the AktDofs, RT-qPCR was conducted on a Thermal Cycler CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) together with PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China). Each sample included three technical replications.
7
miRNA and Target Gene Expression Analysis
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Small RNA used for miRNA verification was extracted by miRcute miRNA Isolation Kit (Tiangen Biotech, China), and first-strand cDNA fragments were produced using miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, China). Total RNA used for target gene verification was extracted with RNAprep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich) (Tiangen Biotech, China), and the reverse-transcription was performed through PrimeScript™ RT reagent Kit (TaKaRa, China). Then, quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed using TB Green TaKaRa Premix Ex Taq™ II (TaKaRa, China) according to the manufacturer’s specification on an ABI Prism 7500 Sequence Detector (Applied Biosystems, United States). The reaction was carried out under the following procedure: denaturation at 95°C for 15 s and 45 cycles of amplification (95°C for 5 s, 58°C for 30 s, and 72°C for 31 s). Ubiquitin and U6 genes were used as internal references for expression normalization of target genes and miRNAs, respectively. Relative expression levels were calculated by 2–ΔΔCT method. The primers used for expression assessment are listed in Supplementary Table 1 . Three biological replicates were used for each qRT-PCR assay.
8
RNA Extraction and Genotyping of IbMYB1
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The total RNAs of experimental materials used in this paper were extracted using the RNA-prep Pure Plant Plus Kit (Polysaccharides and Polyphenolics-rich; Tiangen Biotech [Beijing] CO. LTD.). The RNA of each sample was reverse-transcribed into cDNA by the premix kit (Tiangen Biotech [Beijing] CO. LTD.). The total DNAs of experimental materials used in this manuscript were isolated using a plant genomic DNA kit (Tian gen, Beijing, China). Genomic PCR was conducted for IbMYB1 and structural genes using the PCR conditions and primers described by Mano et al. (2007) (link). For the analysis of the IbMYB1 genotype in ZZP and XLY, IbMYB1-1 and IbMYB1-2 fragments were amplified using PCR conditions and primers (Supplementary Table 1 ) described by Tanaka et al. (2012) (link).
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