Wt ovation exon module

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The WT-Ovation Exon Module is a laboratory equipment designed for RNA-Seq library preparation. It is used for generating amplified cDNA from small amounts of total RNA. The module provides a streamlined workflow for preparing high-quality libraries from total RNA samples.

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Ovation Exon Module (3 citations)

13 protocols using wt ovation exon module

Transcriptome Analysis of Murine B Cell Subsets

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SW_HEL B cell subpopulations (minimum 16,800 cells) were gated as outlined in Fig. 1 B and sorted directly into TRIzol (Invitrogen), and RNA extraction was performed according to the manufacturer’s instructions. To improve recovery, RNA was precipitated with GlycoBlue (Thermo Fisher Scientific). RNA quality and quantity was determined using a Bioanalyzer 2100 (Agilent Technologies). RNA samples were reverse transcribed, amplified, labeled, and fragmented using Ovation Pico WTA, WT Ovation Exon Module, and Encore Biotin Module (NuGen) and hybridized with a whole-transcript gene array on a Mouse Gene 1.0 ST array chip by the Ramaciotti Centre for Genomics (University of New South Wales). One of four LZhi samples did not pass the quality control data assessment of the microarray (Console) and was therefore excluded from the analysis. Computational analysis of gene expression was performed on GenePattern (data normalization, determination of differential gene expression by LimmaGP, and GSEA preranked by fold-change; Broad Institute). For generation of heat maps, transcripts were excluded if they failed to reach a minimal mean expression of log2(x) ≥4. Cutoffs (fold-change and p-values) are indicated in the relevant figures. Genes displayed in heat maps were hierarchically clustered using Pearson correlation.

Kräutler N.J., Suan D., Butt D., Bourne K., Hermes J.R., Chan T.D., Sundling C., Kaplan W., Schofield P., Jackson J., Basten A., Christ D, & Brink R. (2017). Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells. The Journal of Experimental Medicine, 214(5), 1259-1267.

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Amplification and Labeling of RNA

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2 ng total RNA were used to produce amplified single stranded (ss) cDNA with the Ovation Pico WTA System (NuGEN Technologies). 3 µg of the ss cDNA were used to produce double stranded (ds) cDNA with the WT-Ovation Exon Module (NuGEN Technologies). Target labelling was performed using the One-Color DNA Labelling Kit (Roche NimbleGen, Inc.) with 1 µg ds cDNA as input material. All procedures were performed according to the manufacturer’s recommendations.

Tavares B., Jacinto R., Sampaio P., Pestana S., Pinto A., Vaz A., Roxo-Rosa M., Gardner R., Lopes T., Schilling B., Henry I., Saúde L, & Lopes S.S. (2017). Notch/Her12 signalling modulates, motile/immotile cilia ratio downstream of Foxj1a in zebrafish left-right organizer. eLife, 6, e25165.

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Gene Expression Analysis of Periapical Lesions

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For analysis on Affymetrix GeneChip^™ Mouse Gene 1.1 ST Array (Thermo Fisher Scientific Inc., Waltham, MA), total RNA samples were isolated from periapical lesions in hemimandibles (8 (link)) and were subjected to fragmented/labeled cDNA synthesis using the Ovation Pico WTA System, WT-Ovation Exon Module, and the Encore® Biotin Module (all NuGEN Technologies Inc., San Carlos, CA) following manufacturer’s instructions. Changes in gene expression were considered significant if the detection P value was < 0.05 and the change-folds value was >2.0. The infection effect on gene expression profiles in each strain was assessed by either one-way ANOVA or Fisher’s exact t-test using the Partek Genomic suite V6 (Partek, St. Lois, MO). The expression of Hif1a, Cp (ceruloplasmin), and Caix (calbonic anhydrase 9) was further validated by real-time RT-PCR using the 2^−ΔΔCT method and Student’s t-test. Ywhaz (14-3-3 protein zeta/delta) served as a reference gene. The list of primers is shown in Supplemental Materials.

Sasaki H., Furusho H., Rider D.B., Dobeck J.M., Kuo W.P., Fujimura A., Yoganathan S., Hirai K., Xu S., Sasaki K, & Stashenko P. (2019). Endodontic Infection-induced Inflammation Resembling Osteomyelitis of The Jaws in TLR2/IL-10 Double Knockout Mice. Journal of endodontics, 45(2), 181-188.

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Total RNA Isolation and Microarray Analysis

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Total RNA was isolated from sorted nucleated Ter119⁺ cells derived from pooled BM of three mice from each genotype. Microarray expression analysis was performed from samples of two independent experiments. Quantity and integrity of the RNA was assessed by nanoelectrophoresis using the Pico Lab-on-a-Chip assay for total eukaryotic RNA using Bioanalyzer 2100 (Agilent Technologies). Only samples with high integrity (RNA integrity number >8) were subsequently used in microarray experiments. Microarray expression profiles were obtained using the Affymetrix GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) and the GCS3000 Affymetrix Platform (Affymetrix). Briefly, 10 ng of total RNA from each sample was amplified using the Ovation Pico WTA System (NuGEN Technologies) and sense transcript cDNA (ST-cDNA) was generated using the WT-Ovation Exon Module (NuGEN Technologies). After, ST-cDNA was fragmented and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies), and the biotinylated cDNA was hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. Following hybridization, the array was washed and stained, and finally scanned to generate CEL files for each array. Microarray data have been deposited into the Gene Expression Omnibus (GSE54864).

Farrés J., Llacuna L., Martin-Caballero J., Martínez C., Lozano J.J., Ampurdanés C., López-Contreras A.J., Florensa L., Navarro J., Ottina E., Dantzer F., Schreiber V., Villunger A., Fernández-Capetillo O, & Yélamos J. (2014). PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors. Cell Death and Differentiation, 22(7), 1144-1157.

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RNA Amplification and Microarray Analysis

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Total RNA was amplified and labeled using the WT-Ovation FFPE System V2, WT-Ovation Exon Module, and the Encore Biotin Module (NuGEN, Inc. San Carlos, CA, USA), which enables amplification and target preparation of the low quality RNA from FFPE, LCM samples. Amplifications were performed with 100 ng total RNA input following procedures described in the WT-Ovation FFPE System user guide. Target preparation for Affymetrix Human Exon 1.0 ST Arrays (Affymetrix, Santa Clara, CA, USA) was performed using 5 μg amplified cDNA and the Encore Biotin Module following the manufacturer's recommendations. Hybridization, washing and staining (with GeneChip Fluidics Station 450, Affymetrix), and scanning (with GeneChip Scanner 3000) were performed following manufacturer's protocols. Array scans from this process yielded signal intensities comparable to arrays prepared from high quality RNA from cell lines (data not shown). This method has also been independently reported recently to provide optimal RNA hybridization [13 ].

Masterson L., Thibodeau B.J., Fortier L.E., Geddes T.J., Pruetz B.L., Malhotra R., Keidan R, & Wilson G.D. (2014). Gene Expression Differences Predict Treatment Outcome of Merkel Cell Carcinoma Patients. Journal of Skin Cancer, 2014, 596459.

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Microarray Analysis of ALI Assays

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Example 9

RNA obtained from various time points of air-liquid interface (ALI) assays was amplified using the WT Pico RNA Amplification System (NuGEN Technologies Inc., Catalog #3300-12, 3300-60), the WT-Ovation Exon Module, and the Encore Biotin Module (NuGEN Technologies, Inc.), and hybridized onto GeneChip human Exon 1.0 ST Array (Affymetrix, Inc.) according to the respective manufactures' recommendations. GeneChip operating software was used to process all the Cel files and calculate probe intensity values.

To validate sample quality, probe hybridization ratios were calculated using Affymetrix Expression Console software. The intensity values were log 2-transformed, and imported into the Partek Genomics Suite 6.5 (beta). Exons were summarized to genes and a 1-way ANOVA analysis was performed to identify differentially expressed genes. P values and fold-change were calculated for each analysis.

US09964535B2. Treatment of inflammatory diseases (2018-05-08). The Jackson Laboratory [US]. Inventors: Wa Xian [US].

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Affymetrix Human Exon 1.0 ST Array Protocol

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3 μg of SPIA amplified cDNA was used to do the sense–strand cDNA (ST-cDNA) conversion using the WT- Ovation Exon Module (NuGEN Technologies Inc., San Carlos, CA). 5 μg ST-cDNA was fragmented and labeled with FL-Ovation cDNA Biotin Module V2 kit (NuGEN Technologies Inc., San Carlos, CA) and hybridized using Affymetrix Human Exon 1.0 ST arrays (Affymetrix, Inc., Santa Clara, CA). Microarray hybridization and scanning was performed using GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450, and GeneChip Scanner 3000 7G with Autoloader respectively (Affymetrix, Inc., Santa Clara, CA) [19 (link)].

Yeganeh P.N., Richardson C., Bahrani-Mostafavi Z., Tait D.L, & Mostafavi M.T. (2017). Dysregulation of AKT3 along with a small panel of mRNAs stratifies high-grade serous ovarian cancer from both normal epithelia and benign tumor tissues. Genes & Cancer, 8(11-12), 784-798.

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Transcriptional Profiling of PAX2-Expressing Epithelium

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In order to identify genes expressed in PAX2ⁿ epithelium, expression
arrays were generated from formalin-fixed, laser-capture-micro-dissected (LCM)
PAX2ⁿ SCOUTs and benign control oviductal epithelium. RNAs obtained from the
LCM procedure were amplified using the Ovation FFPE WTA System, WT-Ovation Exon Module and
Encore Biotin Module (NuGEN Technologies) and hybridized onto GeneChip® Human Exon
1.0 ST Arrays. GeneChip operating software was used to process all the Cel files and
calculate probe intensity values. To validate sample quality, hybridization ratios were
calculated using Affymetrix Expression Console software. The intensity values were
log₂-transformed and imported into the Partek Genomics Suite. Exons were
summarized to genes and a 1-way ANOVA was performed to identify differentially expressed
genes. P values and fold-change were calculated for each analysis.
Heatmaps were generated using Pearson’s correlation and Ward’s method with
selected genes based on p value. Pathway analyses were performed using
Gene Set Enrichment Analysis (GSEA) software. Candidate biomarkers were culled from these
arrays and are summarized in Table 1.

Ning G., Bijron J.G., Yamamoto Y., Wang X., Howitt B.E., Herfs M., Yang E., Hong Y., Cornille M., Wu L., Hanamornroongruang S., McKeon F.D., Crum C.P, & Xian W. (2014). The PAX2-null immunophenotype defines multiple lineages with common expression signatures in benign and neoplastic oviductal epithelium. The Journal of pathology, 234(4), 478-487.

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Mouse Gene Expression Profiling via Microarray

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Fragmented and labeled cDNA was generated from 1 ng of total RNA using the Ovation Pico WTA System, the WT-Ovation Exon Module and the Encore Biotin Module (all NuGEN) according to manufacturer's instructions and hybridized to the Affymetrix Mouse Gene 1.0 ST Array. Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000.
Microarray data were normalized by Probe Logarithmic Intensity Error (PLIER) using GeneSpring GX11.0 (Agilent), and differentially expressed genes (fold change difference ≥1.5) were identified using a moderated t-test with a Benjamini and Hochberg multiple testing correction of 0.05. Differentially expressed genes were hierarchically clustered. Gene interaction networks and canonical pathways were analyzed using IPA (www.ingenuity.com). Further functional annotation was carried out using a combination of IPA, Kyoto Encyclopedia of Genes and Genomes (KEGG) and SuperPaths (http://www.genecards.org/info.shtml#pathways_interactions). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3517.

Dulneva A., Lee S., Oliver P.L., Di Gleria K., Kessler B.M., Davies K.E, & Becker E.B. (2015). The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum. Human Molecular Genetics, 24(14), 4114-4125.

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Gene Expression Profiling via Affymetrix Exon Arrays

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Three micrograms of the amplified cDNA from the WT-Ovation Pico amplification step were processed with the WT-Ovation Exon Module in GCAS to produce sense strand ST-cDNA following the manufacturer’s (NuGEN, San Carlos, CA, USA) procedure; 5 μg ST-cDNA was fragmented and labeled with the FL-Ovation™ cDNA Biotin Module using a proprietary two-step fragmentation and labeling process. The first step is a combined chemical and enzymatic fragmentation process that yields single-stranded cDNA products in the base range of 50–100 . In the second step, this fragmented product is labeled via enzymatic attachment of a biotin-labeled nucleotide to the 3-hydroxyl end of the fragmented cDNA generated in the first step. Hybridization, washing, and laser scanning of Affymetrix Human Exon 1.0 ST microarrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Hybridization was performed at 45 °C overnight, followed by washing and staining using FS450 fluidics station. Scanning was carried out using the 7G GCS3000 scanner.

Joehanes R., Zhang X., Huan T., Yao C., Ying S.X., Nguyen Q.T., Demirkale C.Y., Feolo M.L., Sharopova N.R., Sturcke A., Schäffer A.A., Heard-Costa N., Chen H., Liu P.C., Wang R., Woodhouse K.A., Tanriverdi K., Freedman J.E., Raghavachari N., Dupuis J., Johnson A.D., O’Donnell C.J., Levy D, & Munson P.J. (2017). Integrated genome-wide analysis of expression quantitative trait loci aids interpretation of genomic association studies. Genome Biology, 18, 16.

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