Nsolver 3

Manufactured by NanoString

Sourced in United States

NSolver 3.0 is a software application developed by NanoString for the analysis and interpretation of data generated from NanoString's nCounter Analysis System. The software provides tools for processing and visualizing gene expression data obtained from NanoString's proprietary digital technology.

Automatically generated - may contain errors

Lab products found in correlation

Ncounter sprint, by NanoString (4 mentions) Ncounter technology, by NanoString (3 mentions) 2100 bioanalyzer, by Agilent Technologies (3 mentions) Ncounter human v3 mirna panel, by NanoString (3 mentions) Cd33 macs beads, by Miltenyi Biotec (2 mentions) Rneasy micro kit, by Qiagen (2 mentions) Trizol plus rna purification kit, by Thermo Fisher Scientific (2 mentions) Ncounter flex system, by NanoString (2 mentions) Immunology panel version 2, by NanoString (2 mentions) Ncounter system, by NanoString (2 mentions) Ncounter analyzer, by NanoString (2 mentions) Digital analyzer, by NanoString (1 mentions) Ncounter sprint profiler machine, by NanoString (1 mentions) Rnaeasy mini kit, by Qiagen (1 mentions) Whole genome dasl assay, by Illumina (1 mentions) Ncounter car t characterization panel, by NanoString (1 mentions) Direct zol rna miniprep kit, by Zymo Research (1 mentions) Rnalater, by Thermo Fisher Scientific (1 mentions) Agilent bioanalyzer, by Agilent Technologies (1 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

NSolver analysis software version 3.0 (15 citations) NSolver Analysis Software 3.0 (11 citations) NSolver 3.0 Analysis Software (5 citations) NSolver 3.0 digital (2 citations) NSolver version 3.0 software (2 citations)

29 protocols using nsolver 3

Comparing Gene Expression Profiles of Ex Vivo and Reactivated Tumor-Infiltrating Lymphocytes

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Example 8

The eTIL and rTIL were assessed identify similarities and/or differences in the gene expression profile of rTIL and eTIL.

In the study, Nanostring's nCounter technology was utilized, which employs a color-coded barcode multiplexed to mRNA to deliver a digital readout of gene expression. Purified RNA (RNeasy, Qiagen) from six matched eTIL and rTIL samples were hybridized with an nCounter Immunology V2 panel codeset for 16 hours on a thermocycler. Codesets consist of a mixture of capture and reporter probes that are multiplexed with the target RNA through 22 bp interactions during thermocycling. Samples were loaded into a 12-well SPRINT cartridge and ran on an nCounter SPRINT device. Count data are exported in a custom RCC format and matched to an RLF file which matches gene names to probe IDs. Normalization and analysis were done on nSolver 3.0 (NanoString Technologies, Inc.).

The results of this study are illustrated in FIGS. 12 and 13. As shown in FIGS. 12 and 13, the gene expression profile is significantly different when comparing the eTIL and rTIL (see the heat map in FIG. 12). There are several genes that are significantly upregulated or downregulated in the rTIL compared to the eTIL (FIG. 13).

US11401507B2. Remnant tumor infiltrating lymphocytes and methods of preparing and using the same (2022-08-02). Iovance Biotherapeutics, Inc. [US]. Inventors: Michelle R. Simpson-Abelson [US], Christopher Mosychuk [US], Michael T. Lotze [US].

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Comparative Gene Expression Analysis of Ex Vivo and In Vivo Expanded TILs

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Example 8

The eTIL and rTIL were assessed identify similarities and/or differences in the gene expression profile of rTIL and eTIL.

US11293009B2. Remnant tumor infiltrating lymphocytes and methods of preparing and using the same (2022-04-05). Iovance Biotherapeutics, Inc. [US]. Inventors: Michelle R. Simpson-Abelson [US], Christopher Mosychuk [US], Michael T. Lotze [US].

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Nanostring miRNA Expression Analysis

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Extracted RNA quality and quantity were analyzed on an agilent Bioanalyzer 2100 using a nano chip. miRNA was hybridized using the Nanostring nCounter® Human v3A miRNA Expression Assay, according to the manufacturer’s protocol. A total of 100 ng RNA were used per sample. Hybridizations were processed on the nCounter Prep Station, and prepped cartridges were scanned on the Nanostring Digital Analyzer using 280 field of view counts. Data were analyzed with Nanostring nSolver 3.0. Samples were analyzed using the default quality control measures, and normalized to ligation controls in the Nanostring codesets.

Snuderl M., Kannan K., Pfaff E., Wang S., Stafford J.M., Serrano J., Heguy A., Ray K., Faustin A., Aminova O., Dolgalev I., Stapleton S.L., Zagzag D., Chiriboga L., Gardner S.L., Wisoff J.H., Golfinos J.G., Capper D., Hovestadt V., Rosenblum M.K., Placantonakis D.G., LeBoeuf S.E., Papagiannakopoulos T.Y., Chavez L., Ahsan S., Eberhart C.G., Pfister S.M., Jones D.T, & Karajannis M.A. (2018). Recurrent homozygous deletion of DROSHA and microduplication of PDE4DIP in pineoblastoma. Nature Communications, 9, 2868.

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Nanostring-based Gene Expression Analysis

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Total RNA from sorted T cell populations (n=4-12 mice/group) was extracted with mini RNA-easy Kit (Qiagen). Equal amount of total RNA from different cells was used for the assay. Hybridization reaction was established by following the instruction of manufacture. Aliquots of Reporter Codeset and Capture probeset were thawed at room temperature. Then a master of mix was created by adding 70ul of hybridization buffer to the tube containing the reporter codeset. 8 μl of this master mix was added to each of tubes for different samples, 5 μl (50 ng) of total RNA sample was added into each tube. Then 2 μl of well mixed Capture probeset was added to each tube and placed in the preheated 65°C thermal cycler. All the sample mixes were incubated for 16 hours at 65°C for completion of hybridization. The samples were then loaded into the sample hole in the cartridge and loaded into the NanoString nCounter SPRINT Profiler machine (NanoString). When corresponding RLF running is finished, the raw data was downloaded and analyzed with Nanostring software nSolver 3.0 (Nanostring). mRNA counts were processed to account for hybridization efficiency, background noise, and sample content, and were normalized using the geometric mean of housekeeping genes. Fold changes were calculated comparing the experimental group to their appropriate controls.

Li C., Zhu B., Son Y., Wang Z., Jiang L., Xiang M., Ye Z., Beckermann K.E., Wu Y., Jenkins J., Siska P.J., Vincent B.G., Prakash Y.S., Peikert T., Edelson B.T., Taneja R., Kaplan M.H., Rathmell J.C., Dong H., Hitosugi T, & Sun J. (2019). The transcription factor Bhlhe40 programs mitochondrial regulation of resident CD8+ T cell fitness and functionality. Immunity, 51(3), 491-507.e7.

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Gene Expression Analysis with nCounter

Cited 1 time

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Expression analysis was performed from 100 ng total RNA using the nCounter system Gene 1 (NanoString Technologies, Seattle, USA). A customized codeset comprising 48 target genes including BDNF and 7 reference genes was hybridized as recommended by the manufacturer. Background correction and normalization of data was performed using the NanoString software nSolver 3.0 (NanoString Technologies). Stably expressed reference genes were chosen for normalization based on the geNorm method, a popular algorithm to determine the most stable reference genes from a set of tested candidate reference genes in a given sample panel. This algorithm calculates a gene expression normalization factor for each sample based on the geometric mean of a user-defined number of reference genes. The underlying principles and formulas are described in (28 (link)). Following this, the selected reference genes were GAPDH, RPS17, TBP and UBC.

Konturek T.J., Martinez C., Niesler B., van der Voort I., Mönnikes H., Stengel A, & Goebel-Stengel M. (2021). The Role of Brain-Derived Neurotrophic Factor in Irritable Bowel Syndrome. Frontiers in Psychiatry, 11, 531385.

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Transcriptomic Profiling of HER2-Positive and Negative Breast Cancers

Cited 1 time

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Two control groups of ER-positive carcinomas, N=22 HER2-negative (IHC score 0/HER2-non-amplified) and N=22 HER2-positive (HER2 IHC score 3+/HER2-amplified), were subjected to global transcriptomics by Whole-Genome DASL assay (Illumina Inc., San Diego, CA) according to the manufacturer’s instructions (Supplementary Methods and related Supplementary Figures, Supplemental Digital Content 1, http://links.lww.com/PAS/A652). Genes with differential expression in HER2-positive versus HER2-negative carcinomas were identified on the basis of t test significance P<0.01 and on mean gene expression variations >±2-fold. Cluster analysis was performed using GEDAS software and the “Fuzzy Self-organizing Maps” algorithm with cosenic distance.17 (link)
Subsequently, these 2 cohorts and the double-equivocal carcinomas confirmed at least by 2 FISH observers were analyzed by a customized nCounter GX CodeSet assay (NanoString) including the gene signature obtained by DASL, 4 housekeeping genes, 6 positive quality controls, and 8 negative quality controls (Supplementary Methods, Supplemental Digital Content 1, http://links.lww.com/PAS/A652). Analysis of genes significantly differentially expressed between subgroups was performed in MeV 4.8 software (version 10.2) using the t test (critical P-value=0.05). Unsupervised clustering was performed by nSolver 3.0 (NanoString).

Marchiò C., Dell’Orto P., Annaratone L., Geyer F.C., Venesio T., Berrino E., Verdun di Cantogno L., Garofoli A., Rangel N., Casorzo L., dell’Aglio C., Gugliotta P., Trisolini E., Beano A., Pietribiasi F., Orlassino R., Cassoni P., Pich A., Montemurro F., Mottolese M., Vincent-Salomon A., Penault-Llorca F., Medico E., Ng C.K., Viale G, & Sapino A. (2018). The Dilemma of HER2 Double-equivocal Breast Carcinomas: Genomic Profiling and Implications for Treatment. The American Journal of Surgical Pathology, 42(9), 1190-1200.

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Targeted circRNA Profiling in FFPE and Plasma

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A custom CodeSet of capture and reporter probes was designed to target regions of 100 nucleotides overlaying the BSJ of 39 selected top candidate circRNAs identified in cohort 1 (Additional file 3: Table S1). In addition, five reference transcripts were included, identified as stably expressed in the total RNA-seq data by the NormFinder algorithm [24 (link)] (ACTB, HPRT1, RPS24, circARHGAP12, and circRBM23).
For FFPE and plasma samples, 300 ng and 5 μL total RNA, respectively, was subjected to nCounter™ SPRINT (NanoString Technologies) analysis according to the manufacturer’s instructions. Hybridization time was set to 23 h. Background subtraction and subsequent normalization was performed using the nSOLVER 3.0 software (NanoString Technologies). A background threshold of 10 was selected, which all raw counts at or below were set to. Normalization was performed using the geometric mean of the five reference transcripts (ACTB, HPRT1, RPS24, circARHGAP12, and circRBM23).

Hansen E.B., Fredsøe J., Okholm T.L., Ulhøi B.P., Klingenberg S., Jensen J.B., Kjems J., Bouchelouche K., Borre M., Damgaard C.K., Pedersen J.S., Kristensen L.S, & Sørensen K.D. (2022). The transcriptional landscape and biomarker potential of circular RNAs in prostate cancer. Genome Medicine, 14, 8.

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Evaluating Tumor Elimination by CAR-T Cells

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WT BV173 cells were co-cultured with either sTCR⁺ or sTCR⁺CoCAR⁺ ATCs at an E:T ratio of 1:5. Four days later, flow cytometry was performed to measure tumor elimination, and 5×10⁵ BV173 cells were added back per well of culture. 24 hours later, the co-cultured cells were harvested. In conditions in which BV173 cells were still present, CD33 MACS beads (130–045-501, Miltenyi Biotech, Germany) were used to deplete residual tumor cells. Total RNA was collected from ATCs using the RNeasy Micro Kit (Qiagen, Germantown, MD) following kit instructions for extraction from cells. Gene expression analysis (NanoString, Seattle, WA) was performed by the Baylor College of Medicine Genomic and RNA Profiling Core using the nCounter CAR-T Characterization Panel. Submitted samples were >250 ng and <5 μL. Data were analyzed using the nSolver 3.0 software (NanoString). See statistical section for additional information on NanoString analysis.

Omer B., Cardenas M.G., Pfeiffer T., Daum R., Huynh M., Sharma S., Nouraee N., Xie C., Tat C., Perconti S., Van Pelt S., Scherer L., DeRenzo C., Shum T., Gottschalk S., Arber C, & Rooney C.M. (2022). A Costimulatory CAR Improves TCR-based Cancer Immunotherapy. Cancer Immunology Research, 10(4), 512-524.

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Profiling Immune Cells in Omentum Tumors

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Omentum tumors were harvested from mice at the time of sacrifice and stored in RNALater. RNA was isolated using Trizol Plus RNA Purification Kit (Invitrogen, cat. # 12183555) according to the manufacturer’s instructions. Aqueous phase solutions were transferred to a spin cartridge found in the PureLink RNA Mini Kit and processed similarly. Samples of high purity (A260/A280 > 2, A260/A230 > 1.4) were processed on the NanoString nCounter Flex system (NanoString Technologies, Seattle, WA, USA) using the mouse PanCan Immune pre-made panels. Briefly, 100 ng of purified RNA was hybridized for at least 16 h with the respective Reporter Code set and Capture Probes for each panel separately. The samples were purified and immobilized on the NanoString Prep Station and counted on the NanoString Digital Analyzer. Analysis was performed using nSolver 3.0 software (NanoString Technologies). Cluster 3.0 and Java TreeView-1.1.6r4 were used to create heat maps. Expression levels were normalized to housekeeping genes that were not discarded by the gNorm program in the advanced analysis module. As stated in the previous section, we utilized NanoString to calculate immune scores for specific immune cell types. Wnt signaling was not calculated for NanoString-only samples as the vast majority of the Wnt signature genes are not measured by NanoString analysis.

Goldsberry W.N., Meza-Perez S., Londoño A.I., Katre A.A., Mott B.T., Roane B.M., Goel N., Wall J.A., Cooper S.J., Norian L.A., Randall T.D., Birrer M.J, & Arend R.C. (2020). Inhibiting WNT Ligand Production for Improved Immune Recognition in the Ovarian Tumor Microenvironment. Cancers, 12(3), 766.

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Profiling of miRNA using nCounter Platform

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The nCounter Human v3 miRNA panel (NanoString Technologies), which targets 799 miRNAs, was used for miRNA profiling according to the manufacturer’s instructions using 100 ng of total RNA from each sample and a hybridization time of 20 h. The hybridization products were then analyzed on the nCounter™ SPRINT (NanoString Technologies) platform. The raw data were processed using the nSOLVER 3.0 software (NanoString Technologies); first, a background subtraction was performed using the max of negative controls, and then positive control normalization was performed using the geometric mean of all positive controls. A second normalization was performed using the geometric mean of the best combination of any two miRs (hsa-let-7d-5p and hsa-miR-423-5p) as determined by the NormFinder algorithm [39 (link)], before exporting the data to Excel (Microsoft Corporation).

Moldovan L.I., Hansen T.B., Venø M.T., Okholm T.L., Andersen T.L., Hager H., Iversen L., Kjems J., Johansen C, & Kristensen L.S. (2019). High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome. BMC Medical Genomics, 12, 174.

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