Ncounter xt assay

Manufactured by NanoString

The NCounter XT Assay is a multiplex gene expression analysis platform that allows for the simultaneous measurement of up to 800 target molecules in a single reaction. The system utilizes color-coded molecular barcodes and digital detection to provide precise quantification of target molecules in a highly multiplexed format.

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Lab products found in correlation

Nanodrop 8000, by Thermo Fisher Scientific (2 mentions) Quick rna miniprep kit, by Zymo Research (2 mentions) Rosettesep cd4 t cell enrichment cocktail, by STEMCELL (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Ficoll plaque plus, by GE Healthcare (1 mentions) Genechip human transcriptome array 2.0 microarrays, by Thermo Fisher Scientific (1 mentions) Ncounter platform, by NanoString (1 mentions) Allprep dna rna mirna universal kit, by Qiagen (1 mentions) Ncounter pancancer immune profiling panel, by NanoString (1 mentions) Tapestation, by Agilent Technologies (1 mentions)

Spelling variants of this product

NCounter assay (30 citations) NCounter XT Codeset Gene Expression Assays (6 citations) NanoString nCounter gene expression assay (XT-CSO-MIP1-12; (2 citations) NanoString nCounter XT CodeSet Gene Expression Assay Protocol (2 citations) NanoString nCounter XT gene expression assay (2 citations)

7 protocols using ncounter xt assay

CD4+ T Cell Transcriptome Analysis in SLE

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CD4⁺ T cells were isolated by negative selection from whole blood using the RosetteSep CD4⁺ T cell enrichment cocktail (Stemcell Technologies) and Ficoll-Plaque Plus (GE Healthcare) by gravity centrifugation. RNA isolated from eight HC and seven SLE patients (one individual with a pair of samples 6 months apart) using the RNeasy Mini Kit (Qiagen) was prepared for GeneChip^™ Human Transcriptome Array 2.0 microarrays (Affymetrix, Thermo Fisher Scientific) with fragmentation, labeling, hybridization and scanning as a single batch. Microarray CEL files were processed by Partek Genomics Suite 6.6 (Partek) using the RMA normalization procedure. Gene set enrichment analysis was carried out with GSEA software [8 (link)] on RMA normalized values using the curated KEGG database v6.0 or HALLMARKS v6.0. A custom gene expression panel (Supplemental Table 1) was designed for nCounter® XT Assay (NanoString Technologies). 50 ng of RNA from CD4⁺ T cell samples were hybridized to cartridges and scanned according to manufacturer instructions. Data was analyzed with nSolver 2.5 with geometric mean normalization to housekeeping genes as well as to positive and negative spike-in controls.

Perry D.J., Titov A.A., Sobel E.S., Brusko T.M, & Morel L. (2020). Immunophenotyping Reveals Distinct Subgroups of Lupus Patients Based on their Activated T Cell Subsets. Clinical immunology (Orlando, Fla.), 221, 108602.

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Nanostring Immune Profiling Protocol

Cited 1 time

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In cohort 1, RNA was purified using AllPrep^®DNA/RNA/miRNA Universal Kit according to manufacturer’s instructions (Qiagen). For multiplex gene expression analysis, the Nanostring nCounter^® platform was used. 100 ng total RNA in 7-20 µl (5-14ng/µl) was used as input. Samples were hybridized to target specific barcodes consisting of a capture probe and a reporter probe using the Nanostring nCounter^® PanCancer Immune Profiling panel (NanoString Technologies Inc, Seattle, WA, USA). After hybridization, target RNA was isolated by magnetic bead sorting and immobilized by applying an electric current as described in the hybridization protocol in the nCounter XT Assay user manual (NanoString Technologies Inc). Gene expression data were normalized with CD8A and CD8B that are stably expressed in CD8⁺ T cells (34 (link)). Two outliers were excluded after nSolver gene expression analysis, why only 38 genotype-selected HS were analysed in cohort 1.

Buhelt S., Laigaard H.M., von Essen M.R., Ullum H., Oturai A., Sellebjerg F, & Søndergaard H.B. (2021). IL2RA Methylation and Gene Expression in Relation to the Multiple Sclerosis-Associated Gene Variant rs2104286 and Soluble IL-2Rα in CD8+ T Cells. Frontiers in Immunology, 12, 676141.

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Lung Transcriptional Profiling of Immune Pathways

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RNA prepared from whole lung tissue was quantified and quality assessed using Tapestation analysis. Gene counts were determined by Peter MacCallum Cancer Centre Advanced Genomic Core by nCounter XT assay (Mouse Pan Cancer Immune Profiling; Nanostring technologies 115000142) and analyzed by nSolver 4.0 software as per manufacturer’s instructions. All positively upregulated genes were tested in ENRICHR tool^{54 (link), 55 (link)}. Top enriched genesets within the Mouse gene atlas^{56 (link)} and Gene Ontology (GO) Biological process 2018 database^{57 (link)} were identified, including macrophage and NK cell genesets^{56 (link)} and Cytokine mediated signaling pathway (GO:0019221). Heatmaps for the top 100 most differentially expressed genes and genes from the cytokine mediated signaling pathway were generated using the pheatmap R package, using row-mean centered and scaled-gene expression of log2(normalized counts + 0.5).

Petley E.V., Koay H.F., Henderson M.A., Sek K., Todd K.L., Keam S.P., Lai J., House I.G., Li J., Zethoven M., Chen A.X., Oliver A.J., Michie J., Freeman A.J., Giuffrida L., Chan J.D., Pizzolla A., Mak J.Y., McCulloch T.R., Souza-Fonseca-Guimaraes F., Kearney C.J., Millen R., Ramsay R.G., Huntington N.D., McCluskey J., Oliaro J., Fairlie D.P., Neeson P.J., Godfrey D.I., Beavis P.A, & Darcy P.K. (2021). MAIT cells regulate NK cell-mediated tumor immunity. Nature Communications, 12, 4746.

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Profiling Stimulated CAR T Cell Transcriptome

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RNA prepared from stimulated CAR T cells was quantified and quality assessed via Tapestation (Agilent). Gene count were determined by the Peter MacCallum Cancer Centre Advanced Genomics Core using an nCounter XT assay (Mouse Pan Cancer Immune Profiling; NanoString Technologies 115000142) and analyzed by nSolver 4.0 software as per the manufacturer’s instructions. Default quality control and normalization steps showed all samples were adequate for further analysis. Heatmaps were generated using the pheatmap R package, using row mean-centered and scaled gene expression of normalized log2 counts derived from nSolver. Rows were grouped by hierarchical clustering using Euclidean distance and average-linkage.

Giuffrida L., Sek K., Henderson M.A., Lai J., Chen A.X., Meyran D., Todd K.L., Petley E.V., Mardiana S., Mølck C., Stewart G.D., Solomon B.J., Parish I.A., Neeson P.J., Harrison S.J., Kats L.M., House I.G., Darcy P.K, & Beavis P.A. (2021). CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nature Communications, 12, 3236.

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Profiling Neuroinflammation and Myeloid Cells

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RNAlater-preserved samples were homogenized in PBS and total RNA was extracted from samples using the Quick-RNA Miniprep Kit (Zymo Research). RNA extracts were evaluated for concentration and purity using a Nanodrop 8000 instrument (Thermo Fisher Scientific) and diluted to a concentration of 20 ng/µl. Hybridizations were performed for the mouse Neuroinflammation, Myeloid cell, and Neuropathology panels according to the nCounter XT Assay user manual (Nanostring). The hybridizations were incubated at 65 °C for 16 h, and then were added to the nCounter SPRINT Cartridge for data collection using the nCounter SPRINT Profiler. Counts were analyzed using the nSolver Analysis Software.

Johnson N.R., Yuan P., Castillo E., Lopez T.P., Yue W., Bond A., Rivera B.M., Sullivan M.C., Hirouchi M., Giles K., Aoyagi A, & Condello C. (2023). CSF1R inhibitors induce a sex-specific resilient microglial phenotype and functional rescue in a tauopathy mouse model. Nature Communications, 14, 118.

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Transcriptional Profiling of Lung Tissue

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RNA prepared from whole lung tissue was quantified and quality assessed using Tapestation analysis. Gene counts were determined by Peter MacCallum Cancer Centre Advanced Genomic Core by nCounter XT assay (Mouse Pan Cancer Immune Profiling; Nanostring technologies 115000142) and analyzed by nSolver 4.0 software as per manufacturer's instructions. All positively upregulated genes were tested in ENRICHR tool (54, 55) . Top enriched genesets within the Mouse gene atlas (56) and Gene Ontology (GO) Biological process 2018 database (57) were identified, including macrophage and NK cell genesets (56) and Cytokine mediated signaling pathway (GO:0019221). Heatmaps for the top 100 most differentially expressed genes and genes from the cytokine mediated signaling pathway were generated using the pheatmap R package, using row mean centered and scaled gene expression of log2(normalized counts+0.5).

Petley E.V., Koay H., Henderson M.A., Sek K., Todd K.L., Keam S.P., Lai J., House I.G., Zethoven M., Chen A., Oliver A.J., Michie J., Freeman A.J., Giuffrida L., Chan J.D., Pizzolla A., Mak J.Y.W., Souza‐Fonseca‐Guimaraes F., Kearney C.J., Millen R., Ramsay R.G., Huntington N.D., McCluskey J., Oliaro J., Fairlie D.P., Neeson P.J., Godfrey D.I., Beavis P.A., & Darcy P.K. (2020). MAIT cells regulate NK cell mediated tumor immunity.

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nCounter-based Neuroinflammation Gene Expression

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RNAlater-preserved samples were homogenized in PBS and total RNA was extracted from samples using the Quick-RNA Miniprep Kit (Zymo Research). RNA extracts were evaluated for concentration and purity using a Nanodrop 8000 instrument (Thermo Fisher Scientific) and diluted to a concentration of 20 ng/µl. Hybridizations were performed for the mouse Neuroinflammation, Myeloid cell, and Neuropathology panels according to the nCounter XT Assay user manual (Nanostring). The hybridizations were incubated at 65°C for 16 hours, and then were added to the nCounter SPRINT Cartridge for data collection using the nCounter SPRINT Profiler. Counts were analyzed using the nSolver Analysis Software.

Johnson N.R., Yuan P., Castillo E., Lopez T., Yue W., Bond A., Rivera B.M., Sullivan M.C., Hirouchi M., Giles K., Aoyagi A., & Condello C. (2021). CSF1R inhibitor levels determine sex-specific phenotype of resilient microglia and neurofunctional rescue leading to extended survival in tauopathy mice.

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