Rnaprep pure plant kit

Manufactured by Tiangen Biotech

Sourced in China, United States, Japan, Germany

The RNAprep Pure Plant Kit is a laboratory equipment product designed for the isolation and purification of total RNA from a variety of plant samples. It utilizes a silica-based membrane technology to efficiently capture and purify RNA molecules.

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Lab products found in correlation

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1 213 protocols using rnaprep pure plant kit

Transcriptome Analysis of Peanut Seed Development

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The developing seeds from early, middle and late stages were collected at 20, 40, and 60 days after owering (DAF) in parents Zhonghua 16 and sd-H1. Three replicates were set for each stage. Total RNA of each seed sample was extracted using RNAprep pure plant kit (DP441, TIANG EN, China). The libraries were sequenced on a HiSeq 4000 (Illumina) to produce paired-end reads with the length of each 150 bp.
The quality of RNA-seq reads were checked using Trimmomatic (Bolger et al., 2014) and the low quality of reads were lter out. Then, Hisat 2 was used to align clean RNA-seq reads to reference genome. The RSEM software (Li and Dewey, 2011) was used to calculate gene expression values. The DESeq2 package (Anders and Huber, 2010) was used for identify differential expressed genes. Total RNA was extracted for qRT-PCR experiment according to the protocol of RNAprep pure plant kit (TIANGEN, China).
The reverse transcription from RNA to cDNA was performed using cDNA Synthesis Kit (TIANGEN, China). Peanut Actin gene was used as the internal control and relative gene expression was calculated by the 2-ΔΔCt method.

Wang Z., Yan L., Chen Y., Wang X., Huai D., Kang Y., Jiang H., Liu K., Lei Y, & Liao B. (2022). Detection of a major QTL and development of KASP markers for seed weight by combining QTL-seq, QTL-mapping and RNA-seq in peanut. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 135(5).

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Transcriptome Analysis of Peanut Seed Development

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Wang Z., Yan L., Chen Y., Wang X., Huai D., Kang Y., Jiang H., Lei Y., & Liao B. (2021). Detection of a Major QTL and Development of KASP Markers for Seed Weight by Combining QTL-seq, QTL-mapping and RNA-seq in Peanut.

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Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted with RNAprep Pure Plant kits (TIANGEN Corporation, Beijing, China) following the manufacturer’s instructions. We used 1 µg of total RNA for first-strand cDNA synthesis. First-strand cDNA was synthesized using the PrimeScript first-strand cDNA synthesis kit (Takara, Dalian, China) or the SMARTer RACE cDNA Amplification Kit (for RACE cloning; Clontech, USA). Synthesized cDNA was subsequently diluted to a final concentration of 20 ng µL⁻¹ with nuclease-free water for use in cloning.

Wang L., Guo Z., Zhang Y., Wang Y., Yang G., Yang L., Wang R, & Xie Z. (2017). Characterization of LhSorP5CS, a gene catalyzing proline synthesis in Oriental hybrid lily Sorbonne: molecular modelling and expression analysis. Botanical Studies, 58, 10.

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Transcriptome Sequencing and Analysis Protocol

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Total RNA was isolated and purified using RNAprep pure Plant Kits (TIANGEN BIOTECH). Approximately 15 μg of total RNA was used for library construction following a standard procedure. Libraries were sequenced with a read length of 100 bp (paired-end) and an insertion size of 300 bp on an Illumina HiSeq 2000 at Berry Genomics, Beijing. Read quality was evaluated using FastQC software (Andrews 2010 ). 3′ reads with quality less than 20 were first trimmed by NGS QC Toolkit (v2.3) (Patel and Jain 2012 (link)). Only reads with a read length greater than 50 bp were kept for downstream analysis. The high-quality reads were then aligned to the B73 reference sequence (AGPv2) (Schnable et al. 2009 (link)) using Tophat2/Bowtie1 (Kim et al. 2013 (link)). Five mismatches, a minimum intron size of 5 bp and a maximum intron size of 60,000 bp were used for alignment. For each sample, the number of reads covering the gene model (filtered gene set 5b) was calculated using htseq-count with the intersection-strict option (Anders et al. 2014 (link)).

Chen Q., Liu Z., Wang B., Wang X., Lai J, & Tian F. (2015). Transcriptome sequencing reveals the roles of transcription factors in modulating genotype by nitrogen interaction in maize. Plant Cell Reports, 34(10), 1761-1771.

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Transcriptome Analysis of Arabidopsis Mutants

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Total RNA was extracted from 4-week-old 862 strain, 35S::ed1 and Pro_ED1::ed1, leaves with two biological replicates using RNA Prep Pure Plant Kits (Tiangen). The libraries were constructed and sequenced using an Illumina HiSEquation 2000 (Benagen). ~ 25 million raw reads from each sample were collected and filtered. Clean reads were mapped to the reference genome (http://www.genoscope.cns.fr/brassicanapus/) using Hisat2. The gene expression levels were calculated by the FPKM method based on the number of uniquely mapped reads. Differential expression analysis was performed with the DESeq2 R package using |log2 (fold change)| ≥ 1 and a corrected P < 0.05 as the threshold for significant differential expression. P values were adjusted using the Benjamini and Hochberg approach to control for false discovery rates. Functional enrichment analyses, including Gene Ontology (GO) and KEGG, were performed on DEGs, which were compared to the whole genome background using the hypergeometric test with Benjamini and Hochberg’s false discovery rate correction at the significance threshold of 0.05.

Zheng M., Hu M., Yang H., Tang M., Zhang L., Liu H., Li X., Liu J., Sun X., Fan S., Zhang J., Terzaghi W., Pu H, & Hua W. (2019). Three BnaIAA7 homologs are involved in auxin/brassinosteroid-mediated plant morphogenesis in rapeseed (Brassica napus L.). Plant Cell Reports, 38(8), 883-897.

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Transcriptome Analysis of Lonicera spp.

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Total RNA from L. japonica and L. macranthoides was extracted from young flower buds and leaves using RNA-prep Pure Plant Kits (Tiangen, China). cDNA for the rapid-amplification of cDNA ends (RACE) was prepared according to the user manual for the SMART RACE cDNA Amplification Kit (Clontech, Japan), and the cDNA for the RT-PCR analyses was synthesized using the AMV Reverse Transcription System (Promega, USA). Genes were amplified by using a Fast HiFidelity PCR Kit (Tiangen, China), according to the manufacturer’s instructions; PCR products were cloned into the pLB vector (Tiangen, China) and sequenced. The sequences of newly found genes from this study were submitted to the GenBank database and the accession numbers are listed in Supplementary Table S1. For sequence alignment and phylogenetic analysis, the deduced amino acid sequences were compared with the MEGA 4.0 program using the Neighbor-Joining method with 1000 bootstrap replications48 (link).

Wu J., Wang X.C., Liu Y., Du H., Shu Q.Y., Su S., Wang L.J., Li S.S, & Wang L.S. (2016). Flavone synthases from Lonicera japonica and L. macranthoides reveal differential flavone accumulation. Scientific Reports, 6, 19245.

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Canola Seed Total RNA Isolation and qPCR Analysis

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Total RNA was isolated using RNAprep pure Plant Kits (Tiangen, Beijing, China) from canola seeds at 2, 4, 6, and 8 WAP. RNA samples were quantified by NanoDrop 2000c Spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and their integrity and purity confirmed using agarose gel electrophoresis. 1 μg of total RNA was reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad, USA) according to the manufacturer’s protocol. PCR primers were designed with Primer 5.0 and Oligo 6.0 software. All RT-PCR reactions were performed using iTaq^TM Universal SYBR^® Green Supermix (Bio-Rad, USA) in a CFX96^TM Real-Time PCR Detection System. The qPCR cycling conditions were as follows: one cycle of 95°C for 30 s, then 39 cycles of 95°C for 5 s and 55–70°C for 1 min, followed by a melting curve ramping from 65 to 95°C with temperature increasing by 0.5°C every 5 s (one cycle). Data were analyzed using the 2^-ΔΔCT method (Livak and Schmittgen, 2001 (link)), normalized to beta-actin. Three replicates were used for each gene and data were analyzed using CFX Manager^TM v3.0.

Wan H., Cui Y., Ding Y., Mei J., Dong H., Zhang W., Wu S., Liang Y., Zhang C., Li J., Xiong Q, & Qian W. (2017). Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola. Frontiers in Plant Science, 7, 2007.

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Quantitative RT-PCR of Rapeseed Genes

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Total RNA was extracted using RNA Prep Pure Plant Kits (Tiangen). The cDNA was synthesized using the First cDNA Transcriptase and Oligo (dT)18 primer (Takara). qRT-PCR was performed using a Fast Start Universal Probe Master Mix (Roche) in an ABI 7500 Fast PCR system with three biological replicates. The rapeseed TMA7 gene (BnaC05g11560D) was used as control. Primers for qRT-PCR are listed in Supplemental Table 6. Data were analyzed following the relative quantification method (

2^{- Δ Δ C_{T}}

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Whole-Genome RNA-seq of Cotton Roots

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For whole-genome RNA-seq, the V. dahliae-inoculated roots of CSSL-1 (RRI) and CSSL-4 (RSI), and the control roots of CSSL-1 (RRM) and CSSL-4 (RSM) were used. Three biological replicates were performed for each treatment.
Total RNA was isolated from each cotton seedling sample using RNAprep Pure Plant Kits (TIANGEN Biotech, Beijing, China) . Ribosomal RNA was removed using the Ribo-Zero Gold Kit (Epicentre, Madison, WI, USA). Sequencing libraries were constructed using the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), according to the manufacturer's instructions. The libraries were sequenced on the Illumina HiSeq 4000 platform and 150 bp paired-end reads were generated.

Zhang L., Liu J., Cheng J., Sun Q., Zhang Y., Liu J., Li H., Zhang Z., Wang P., Cai C., Chu Z., Zhang X., Yuan Y., Shi Y, & Cai Y. (2022). lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt. Plant physiology, 189(1).

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Quantitative Real-Time PCR Analysis of BCCP Subunit Expression in A. moluccana Endosperm

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Total RNA was isolated from the A. moluccana endosperm using RNAprep Pure Plant kits (TIANGEN Biotechnology Company, Beijing, China) according to the manufacturer protocol. First strand cDNA was synthesized using the RevertAid-TM First Strand cDNA synthesis kit (Fermentas) according to the manufacturer protocol. qRT-PCR was performed to examine expression of the BCCP subunit gene. The 18SrRNA gene was used as an internal control in A. moluccana. The specific primers for the internal control gene and the target gene used for qRT-PCR are shown in Table 1. The amplified fragment lengths of the control and target gene were 110 bp and 217 bp, respectively. Finally, cDNA was amplified using real-time PCR. All amplifications were performed using SYBR Premix ExTaq II (TaKaRa Biotechnology Company, Dalian, China). SYBR Green II assay reactions were performed in a 10 μL volume and each reaction contained 5 μL SYBR Premix ExTaq II, 400 nM concentration of each primer, and cDNA. PCRs were performed with four biological replicates. Gene expression levels were evaluated using the delta/delta calculation method (Livak and Schmittgen, 2001) . The data were then processed using the base 10 logarithm and Excel mapping.

Xuan W.Y., Zhang Y., Liu Z.Q., Feng D, & Luo M.Y. (2015). Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana. Genetics and molecular research : GMR, 14(3).

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