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Omega plant rna kit

Manufactured by Omega Bio-Tek
Sourced in United States, China

The Omega Plant RNA kit is a laboratory product designed for the extraction and purification of high-quality total RNA from a variety of plant tissues. It utilizes a guanidinium-based lysis solution and silica-based membrane technology to efficiently capture and isolate RNA molecules.

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20 protocols using omega plant rna kit

1

Quantitative Real-Time PCR Analysis of Gene Expression

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Total RNA from the samples under different stress treatment was extracted using the OMEGA plant RNA Kit (Omega Bio-Tek, Shanghai, China) and following the operating instruction. cDNA synthesis was followed by Takara’s PrimeScript Synthesis 1st Strand cDNA Synthesis Kit (Takara, Beijing, China). Quantitative real-time PCR analysis was performed with TransStart ®Top Green qPCR SuperMix (Transgen, Beijing, China) and a Bio-Rad CFX-96 detection system, as follows: 94 °C for 30 s, then 45 cycles of 94 °C for 5 s, 60 °C for 1 s. β-actin was used as a reference gene. All primers used for q-PCR were listed in Table S2. The raw Ct data were analyzed by 2−∆∆Ct [59 (link)]. Three technical replicates were performed for each three independent biological replicates.
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2

RNA-Seq Analysis of Seed Germination

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Total RNA was respectively isolated from the seeds of the four germination stages using the Omega Plant RNA kit (No.R6827, Omega Bio-Tek Inc., USA). RNA purity and concentration of all samples were determined using the Nanodrop 2000 spectrophotometer and QUBIT uorometer (Life Technologies).
RNA integrity was determined using the Agilent 2100 bioanalyzer (Agilent Technologies). High-quality RNA with an RNA integrity number (RIN) > 8 and of su cient quantity was used to construct the sequencing library. RNA samples were used for poly(A) + selection using oligo (dT) magnetic beads. Then, libraries were sequenced using the Illumina HiSeq TM 4000 platform (Illumina, San Diego, CA, USA) at Gene Denovo Technology Co. Ltd., Guangzhou, China. Raw data of RNA-Seq were collected, and clean data were obtained by removing adapters, unknown nucleotides, and low-quality (Q-value ≤ 10) bases. The Q20, Q30, GC content, and sequence duplication levels of the clean data were simultaneously calculated. High-quality clean data were used for downstream analyses. The clean reads were subsequently de novo assembled using the Trinity software (trinityrnaseq-2.0.6) [61] .
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3

Wheat Homoeolog Expression Patterns

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The expression levels of TaAGOs in seedlings, leaves, roots, stems, spikes, grains, stamens, and pistils were downloaded as Transcripts Per Million (TPM) from http://www.wheat-expression.com. A homoeolog expression pattern analysis for triads (a single gene copy per subgenome, i.e., A : B : D configuration of 1 : 1 : 1) with a total expression above 0.5 TPM was conducted. Homoeolog expression bias was classified as balanced (similar relative transcript abundance from A, B, and D homoeologs), A/B/D-suppressed (lower abundance of transcripts from A/B/D homoeologs), or A/B/D-dominant (higher abundance of transcripts from A/B/D homoeologs) patterns as described previously [28 (link)]. The ternary plot was generated using the R package “vcd.” Chinese Spring (Triticum aestivum L.) plants were grown in a growth chamber under 16 h/8 h light/dark period until anthesis stage. Flag leaves were dissected after 6 and 24 h of heat (42°C), PEG-6000 (25%), and NaCl (200 mM) treatment. Total RNA was extracted with Omega plant RNA kit (Omega Bio-tek, China). cDNA was synthesized using EasyScript (Trans). Glyceraldehyde3-phoshate dehydrogenase (GAPDH) was used as normalization control. Relative expression levels were calculated with the 2-ΔΔCt method. The primer sequences used for qRT-PCR are listed in Supplementary Table 1.
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4

Quantification of WRKY Gene Expression

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The total RNA was extracted with the Omega plant RNA kit (Omega Bio-Tek, Georgia, USA) according to the instructions provided by the manufacturer. A total of 1 μg of RNA was reverse-transcribed into cDNA using PrimeScript RT reagent with the gDNA Eraser kit (TaKaRa, Dalian, China). A control amplicon was generated using the β-actin primers for amplification of β-actin (S1 Table). The primers of O. officinalis WRKY genes were designed by the online tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) (S1 Table). Gene expression levels were determined by perfoming quantitative real-time polymerase chain reaction (qRT-PCR) in Applied Biosystems QuantStudio 6 Flex (ABI, USA) using SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. Datas were analyzed by QuantStudio 6 Flex software (ABI, USA) and the 2-△△CT method [29 ].
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5

Transcriptome Analysis of Camellia sinensis

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Purple young leaves (one bud with two leaves) and green mature leaves of ‘Zijuan’ (ZJ) and young leaves (one bud with two leaves) of ‘Yunkan-10#’ (YK) [both cultivars of Camellia sinensis var. asssamica (Masters) Kitamura] were sampled, with six replications, from healthy plants grown in the tea garden of the Tea Seed Multiplication Farm of Pu’er City, Yunnan Province, China. All samples were collected in the morning in early August, quickly frozen in liquid nitrogen, and stored at 80° until total RNA extraction.
Total RNA was extracted using an Omega Plant RNA kit (Omega Bio-Tek, Code: R6827) according to the manufacturer’s protocol. The quality, integrity and quantity of total RNA were assessed using a NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific, Wilmington, DE, USA) and an Agilent Bioanalyser 2100 with the Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA), respectively.
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6

C. arbutifolia Leaf Extraction Protocol

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Branches from a superior individual of C. arbutifolia were collected in the town of Manjiang (41°47′10.55″, 127°55′56.13″), Fusong County, Jilin Province, China. All branches were transported back to the laboratory of the Jiangsu Academy of Forestry for hydroponic cultivation until leaves had sprouted. A DNA extraction kit (DP305; Tiangen Biotech, Beijing, China) was used to isolate genomic DNA from young leaves of C. arbutifolia. An Omega Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA) was used for total RNA extraction from the leaves of C. arbutifolia.
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7

Quantifying Gene Expression in Apple

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Total RNA of apple branches, N. benthamiana leaves, and C. mali were extracted using the Omega Plant RNA Kit (Omega Bio-Tek), and then, total RNA was reverse-transcribed to cDNA using an EasyScript cDNA Synthesis Kit (Transgene, China). The detection of gene expression levels was performed in triplicate using gene-specific primers with SYBR Green staining. The expression of Actin was used as an internal standard for RNA samples.
The species specificity of primers used for analyzing the gene expression of C. mali in apple calli was determined by PCR and gel electrophoresis (Supplemental Fig. S17). The RNA of infected apple branches and calli with high- and low-K status were extracted and reverse-transcribed to cDNA. The pathogen gene expression was determined in triplicate using these gene-specific primers by RT-qPCR. The expression of Actin was used as an internal standard for RNA samples. The data of RT-qPCR were analyzed through the 2−△△CT method.
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8

RNA-seq Analysis of Plant Transcriptome

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Total RNA from leaves was extracted using the Omega Plant RNA kit (Cat. no. R6827–01, Omega Bio-tek, Doraville, GA, USA) following the manufacturer’s instructions. RNA–seq libraries were constructed using the Illumina TruSeq RNA Kit (NEB, Cat. no. E7530) and were sequenced with an Illumina NovaSeq 6000 system (150 bp paired–end mode). Two biological replicates were performed for RNA–seq experiment. RNA–seq sequencing reads were cleaned as described above. The clean reads were then mapped to the reference genome using Tophat2 v.2.1.1 [51 (link)] with default settings. The Cufflinks v.2.2.1 [52 (link)] was employed to calculate the gene expression levels represented by FPKM (fragments per kilobase of transcript per million mapped reads) values. To compare expression levels across samples and genes, FeatureCounts v.2.0.1 [53 (link)] was used to calculate the read counts per gene and differential expression analysis was performed using DEseq2 [54 (link)] based on threshold criteria of an adjusted p–value  < 0.05 and |log2(fold change)|  >  1. Gene ontology (GO) enrichment analysis was performed by using an online resource (accessed on 24 December 2022, www.omicshare.com/tools) with default instructions.
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9

Transcriptome Profiling of GM Maize

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Four MhSnf7 GM (5–6 leaf stage) and four non-GM maize plants (5–6 leaf stage) were randomly selected to serve as four biological replicates. Total RNA from maize leaves was extracted using Omega Plant RNA kit (Omega Bio-Tek, USA) and analyzed by agarose gel electrophoresis. Poly-oligo dT magnetic beads were used to extract mRNA from total RNA. The transcriptome sequence library was constructed using the NEBNext® Ultra™ RNA LibraryPrep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions. The RNA-seq library was sequenced on the Illumina Hiseq 2000 platform. Clean reads were obtained by removing adapter sequences and omitting reads containing more than 10% unknown nucleotides or with low quality (containing more than 50% bases with Q-score ≤ 20).
Gene expression levels were evaluated in fragments per kilobase million (FPKM) based on the number of fragments mapped to the reference sequence. Differential expression analysis was performed using the DEGseq R package. Genes with an adjusted false discovery rate (FDR) < 0.05 and |log2 fold change|> 1 were scored as differentially expressed.
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10

Wheat Gene Expression Analysis by qPCR

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Total RNA was extracted from wheat shoots and roots of the control and WV treated groups using an OMEGA plant RNA kit (R6827, Omega Bio-tek, Norcross, GA, USA), and cDNA was reverse transcribed from 1 μg of total RNA using the GoScript™ Reverse Transcription System (A5001, Promega, Madison, WI, USA). Relative quantification of gene expression by qPCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Singapore, Singapore). The primers used for qPCR were designed using the qPrimerDB database [76 (link)], Oligo 7 and Beacon Designer™ 8.0 software. The sequence of the primers can be found in the Supplementary Table S3. Wheat actin gene was used as the endogenous control which remained stable throughout the drought treatment [77 (link),78 (link)]. qPCR was performed in an optical 96-well plate, including 10 μL 2 × GoTaq® qPCR Master Mix (A6002, Promega, Madison, WI, USA), 2 μL 1:5-diluted template cDNA, and 0.2 μM of each gene-specific primer, in a final volume of 20 μL, using the following thermal cycles: 95 °C for 1 min, 40 cycles of 95 °C for 10 s, 60 °C for 1 min. Disassociation curve analysis was performed as follows: 95 °C for 15 s, 60 °C for 1min, and 95 °C for 15 min. Relative expression levels were calculated by the 2−ΔΔCt method [79 (link)].
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