Nsolver analysis software v4

Manufactured by NanoString

Sourced in United States

NSolver Analysis Software v4.0 is a data analysis tool developed by NanoString. The software is designed to process and analyze data generated from NanoString's nCounter Analysis System. It provides users with the necessary functionality to perform quality control, normalization, and statistical analysis on their experimental data.

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26 protocols using nsolver analysis software v4

nSolver Analysis for GBM Transcriptome

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The nSolver™ Analysis Software v4.0 (NanoString Technologies®) was applied to evaluate quality control parameters such as binding density, the limit of detection, and positive controls. In addition, the raw data were evaluated in the R statistical environment (version 3.6.3) with the Quantro package (version 1.18.0) to guide the normalization process. No quality control flags were detected, and all 99 samples were included in further analysis. Housekeeping selection, normalization, differential expression, and immune-oncology-related scores calculation were performed in the Advanced Analysis module from the nSolver™ Analysis Software v4.0 (NanoString Technologies®).
The geNorm algorithm implemented in the advanced analysis module was used for the automatic selection of the housekeeping genes for data normalization. The cutoff to consider differentially expressed genes was the adjusted p value less than 0.1. Further statistical analyses of the pathway and cell type scores were performed using the nonparametric Mann–Whitney test for independent samples (IBM SPSS 2.3 version).
Pathway images were generated using the Kyoto Encyclopedia of Genes and Genomes (KEGG), available in the nSolver Advanced Analysis module. Normalized data of differentially gene expression data were downloaded from the nSolver software for survival analysis of IDH^wt GBM.

Moreno D.A., da Silva L.S., Gomes I., Leal L.F., Berardinelli G.N., Gonçalves G.M., Pereira C.A., Santana I.V., Matsushita M.D., Bhat K., Lawler S, & Reis R.M. (2022). Cancer immune profiling unveils biomarkers, immunological pathways, and cell type score associated with glioblastoma patients’ survival. Therapeutic Advances in Medical Oncology, 14, 17588359221127678.

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TCR Sequencing and Immune Profiling of Tumor Samples

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Genomic DNA was extracted from tumors using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). TCRβ sequencing was performed by Adaptive Biotechnologies (Seattle, WA), and analyzed using the immunoSEQ analyzer software by Adaptive Biotechnologies (Adaptive Biotechnologies).
Total RNA was extracted from the indicated tumors on day 28 post tumor transplant using the RNeasy Mini kit (Qiagen, Germantown, MD). nCounter PanCancer Immune Profiling Panel (NanoString Technologies; Seattle, WA) analysis, run by the Genomics Laboratory, Frederick National Laboratory for Cancer Research (Frederick, MD), was performed on the RNA. Data files were analyzed using the nSolver analysis software v.4.0.70 (NanoString; Seattle, WA) with the mRNA counts normalized to housekeeping genes and with the untreated sample as the categorical reference value. Nanostring data are available in the GEO database under accession number GSE162799. Common genes associated with immune activation, suppression, and exhaustion were depicted.

Fabian K.P., Malamas A.S., Padget M.R., Solocinski K., Wolfson B., Fujii R., Sater H.A., Schlom J, & Hodge J.W. (2020). Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable. Cancer immunology research, 9(2), 239-252.

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Nanostring-based Immune Profiling of Tumor Cells

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Total RNA was extracted from the tumor cells or tumor explants using the RNeasy Mini kit (Qiagen) and was analyzed using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies), run by the Genomics Laboratory, Frederick National Laboratory for Cancer Research. The nSolver analysis software V.4.0.70 (NanoString) was used to evaluate the data using housekeeping genes as the normalizing controls and the untreated samples as the categorical reference value.

Fabian K.P., Padget M.R., Fujii R., Schlom J, & Hodge J.W. (2021). Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve. Journal for Immunotherapy of Cancer, 9(2), e001691.

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RNA Isolation and Gene Expression Analysis

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RNA was isolated from the NZM cells using the RNAqueous™ total RNA isolation kit (cat# AM1931, Invitrogen, ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol. The RNA quality was assessed using the Agilent TapeStation. RNA samples were assessed for expression of all genes detailed in Table A2, by using the NanoString nCounter^® (Seattle, WA, USA). The procedure was conducted by Jason Capedo, of Auckland Clinical Genomics (University of Auckland). The nCounter output of raw mRNA barcode counts was analysed using the nSolver™ Analysis Software v4.0 (NanoString). Herein, the raw mRNA barcode counts were adjusted against background and three housekeeper genes to give the absolute mRNA barcode count for the gene of interest.

Anchan A., Martin O., Hucklesby J.J., Finlay G., Johnson R.H., Robilliard L.D., O’Carroll S.J., Angel C.E, & Graham E.S. (2020). Analysis of Melanoma Secretome for Factors That Directly Disrupt the Barrier Integrity of Brain Endothelial Cells. International Journal of Molecular Sciences, 21(21), 8193.

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Gene Expression Analysis of M1/M2 Macrophages

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Gene expression in each sample was assessed using NanoString nCounter Analysis System (NanoString Technologies), as previously described, using a custom-designed codeset containing 280 genes (NanoString Technologies) according to the manufacturer’s instructions. Each reaction contained 150 ng of total RNA, plus reporter and capture probes, together with six pairs of positive control probes and eight pairs of negative control probes. Raw NanoString data were analyzed and normalized using nSolver Analysis Software v4.0 (NanoString Technologies). Counts associated with genes were normalized to internal levels of the reference genes, TNF-α, IL-6, IL-1β, and CCL2 (for M1 macrophages) and TGF-β1, IL-10, IL-11, and Arg1 (for M1 macrophages).

Lee B.M., Park S.J., Noh I, & Kim C.H. (2021). The effects of the molecular weights of hyaluronic acid on the immune responses. Biomaterials Research, 25, 27.

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Circulating miRNA Expression Change

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Normal distribution was tested using the Shapiro–Wilk test. Log2 transformed data by nSolver Analysis Software v. 4.0 (NanoString Technologies) was used to analyze the expression of each miRNA with the visual genomics analysis studio (VGAS) program (http://www.iiid.com.au/software/vgas). Comparison of the pre- and post-vitamin C supplementation data was performed using a paired t-test. Laboratory measures and values from the validation study were compared between pre-and post-supplementation by paired t-test and Wilcoxon signed ranks test using SPSS statistics v. 19 (SPSS Inc., Chicago, IL, USA). A power of test (1-β) was checked by post-hoc analysis using G*Power v. 3.1.9.2 (Heinrich-Heine-Universität Düsseldorf, Germany). Spearman correlation was used to analyze the correlation between circulating miRNA expression and blood parameters and the figures were generated using GraphPad Prism v. 5.0 (GraphPad Software Inc., CA, USA). A p-value of <0.05 was set as the significance threshold. All data expressed as mean ± SD.

Ruknarong L., Boonthongkaew C., Chuangchot N., Jumnainsong A., Leelayuwat N., Jusakul A., Gaudieri S, & Leelayuwat C. (2021). Vitamin C supplementation reduces expression of circulating miR-451a in subjects with poorly controlled type 2 diabetes mellitus and high oxidative stress. PeerJ, 9, e10776.

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Irradiation Impacts on Tumor Gene Expression

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A select group of B16 tumors, received 1 × 11, 1 × 15, 3 × 6 or 3 × 8 Gy, were harvested 96 h postirradiation for mRNA-based (Nanostring Technologies) genetic assessment and CD8+/CD4− immunohistochemistry. RNA isolation and purification was performed immediately after euthanasia using the Qiagen RNeasy Mini Kit. mRNA expression was quantified via the NanoString PanCancer IO 360 (murine) panel. This panel quantifies the expression of 600 genes involved in 28 cancer-related cellular pathways, including tumor growth, tumor microenvironment, and immune response. Expression values were analyzed with the Nanostring nSolver Analysis software (v. 4.0) and Advanced Analysis Software (v. 2.0).

Matsuda Y., Hirobe T, & Chapman V.M. (1991). Genetic basis of X-Y chromosome dissociation and male sterility in interspecific hybrids.. Proceedings of the National Academy of Sciences of the United States of America, 88(11), 4850-4854.

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Detecting Gene Fusions in Lung Cancer

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Validation of the presence of ALK, RET, and ROS1 rearrangements was performed using the Lung Fusion assay (NanoString Technologies). This assay was designed for the evaluation of transcripts using specific probes for the 5' and 3' regions from ALK, RET, and ROS1 genes and specific probes for the rearrangement partners from ALK, RET, ROS1 and NTRK1 (no NTRK imbalance probes are provided in this assay). The transcription counts were normalized by the nSolver Analysis^® Software v4.0 (NanoString Technologies). The calculation of the imbalance between the 3' and 5' probes was performed by a t-test comparing the log-scale data from probes. A significant P value provided the final positive result about fusion presence from the t-test plus the detection of fusion partners. These analyses were conducted using the Advanced Analysis v2.0 package (NanoString Technologies).

Novaes L.A., Sussuchi da Silva L., De Marchi P., Cavagna R.D., de Paula F.E., Zanon M.F., Evangelista A.F., Albino da Silva E.C., Duval da Silva V., Leal L.F, & Reis R.M. (2021). Simultaneous analysis of ALK, RET, and ROS1 gene fusions by NanoString in Brazilian lung adenocarcinoma patients. Translational Lung Cancer Research, 10(1), 292-303.

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Profiling Gene Expression in Macrophages

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Gene expression in each sample was assessed using NanoString nCounter Analysis System (NanoString Technologies), as previously described, using a custom-designed codeset containing 280 genes (NanoString Technologies) according to the manufacturer's instructions. Each reaction contained 150 ng of total RNA, plus reporter and capture probes, together with six pairs of positive control probes and eight pairs of negative control probes. Raw NanoString data were analyzed and normalized using nSolver Analysis Software v4.0 (NanoString Technologies). Counts associated with genes were normalized to internal levels of the reference genes, TNF-α, IL-6, IL-1β, and CCL2 (for M1 macrophages) and TGF-β1, IL-10, IL-11, and Arg1 (for M1 macrophages).

Lee B.M., Park S.J., Noh I., & Kim C. (2021). The Effects of the Molecular Weights of Hyaluronic Acid on the Immune Responses.

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Immune and Inflammatory Gene Expression Analysis

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Immune and inflammatory panels were selected for gene expression analysis using NanoString nCounter® Platform (NanoString Technology). The reactions were set up according to the nCounter XT Gene expression protocol. Gene expression analysis was performed using nSolver Analysis Software v4.0 (NanoString Technology) involving in 770 genes using immune panel (NS_CANCERIMMUNE_V1.1) and a custom designed set of 256 genes in inflammatory panel (NS_INFL_HS_V2_C2534). A background correction was made by subtracting the background thresholds of negative control from raw counts. Adjusted raw counts were normalized using the combination of positive controls and reference gene normalization. The positive control normalization was computed by the geometric mean normalization factor, whereas reference gene normalization was calculated by the geometric mean of 11 genes, SF3A3, MRP55, TLK2, HDAC3, PPIA, TBP, ZNF143, DNAJC14, ABCF1 SAP130, and EDC3, in immune panel and four genes, CLTC, CUSB, HPRT1, and PGK1, in inflammatory panel.

Jantaravinid J., Jiamton S., Srisawat C., Suktitipat B, & Tirawanchai N. (2021). Expression Pattern of Genes in Condyloma Acuminata Treated with Clinacanthus nutans Lindau Cream versus Podophyllin. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 5579520.

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