Total RNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany) as described previously [33 (link)]. Genomic DNA contamination was eliminated by digesting the RNA with RNase-free DNase (Ambion, Cambridge, MA, USA). RNA was quantitated using Nanodrop (Thermo Scientific, Waltham, MA, USA), and the 28S/18S RNA ratios of all RNA samples were between 1.8 and 2.0. RNA quality was analyzed using the Bioanalyzer [33 (link),41 (link)]. For miRNA analysis, we used the nCounter® Mouse miRNA Expression Panel (NanoString, Seattle, WA, USA, Cat: CSO-MMIR15-12). Raw data was normalized using the geometric mean values of the top 100 expressed miRNA in each sample using the nSolver Analysis Software (NanoString), according to the manufacturer’s guidelines. For mRNA analysis, we utilized the nCounter® Mouse PanCancer Immune Profiling Panel to count 770 immune-related genes (NanoString, Cat: XT-CSO-MIP1-12). Raw data was normalized with a set of housekeeping genes and analyzed using the nSolver Analysis Software (NanoString), according to the manufacturer’s guidelines.
Nsolver analysis software
Manufactured by NanoString
Sourced in United States
NSolver Analysis Software is a computational platform designed to analyze data generated from NanoString's gene expression profiling systems. The software enables users to process, visualize, and interpret complex biological data in a streamlined and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Ncounter system, by NanoString (16 mentions)
Ncounter analysis system, by NanoString (9 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
Ncounter sprint profiler, by NanoString (7 mentions)
Ncounter digital analyzer, by NanoString (6 mentions)
Ncounter pancancer immune profiling panel, by NanoString (5 mentions)
Ncounter prep station, by NanoString (4 mentions)
Ncounter mouse immunology panel, by NanoString (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Rneasy lipid tissue mini kit, by Qiagen (3 mentions)
Advanced analysis module, by NanoString (3 mentions)
Ncounter mouse inflammation v2 panel, by NanoString (3 mentions)
Mouse pan cancer immune oncology kit, by NanoString (3 mentions)
Liberase, by Roche (3 mentions)
Rlt buffer, by Qiagen (3 mentions)
Mouse pancancer immune profiling panel, by NanoString (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Ncounter mouse pancancer immune profiling panel, by NanoString (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
135 protocols using nsolver analysis software
1
Isolation and Analysis of RNA and miRNA
2
Single-cell RNA profiling of CAR T-cell therapy
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Tumour samples were collected from NXS2-mCD19 animals on day 18 of the experiment. The samples were collected and enzymatically digested (Liberase, Roche) into single-cell suspensions. CD45+ cells were sorted using BD FACS Melody (BD Biosciences) and RNA was isolated from these cells (RNeasy Plus mini kit, Qiagen). Then, mRNA levels were directly measured using the Mouse-Pan cancer immune-oncology kit from NanoString nCounter gene expression system (NanoString). Differential expression analyses of mRNA were performed using nSolver analysis software (NanoString) and visualized by ClustVis39 (link). Gene Ontology (GO) annotation analysis of the target genes was performed using the Metascape tool (http://metascape.org ), which facilitates enrichment analysis of biological processes and pathways of input genes40 (link). Only genes upregulated more than 2 times in the CAR(NAP) T-cell group compared with the CAR T-cell group were imported into Metascape and output P value cut-off was set to P < 0.0001. Cell type profiling analyses were performed using nSolver analysis software (NanoString).
3
RNA Profiling of FFPE Tumor Samples
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Total RNA was isolated from 10 μm FFPE (Formalin-fixed –paraffin-embedded) - tumor sections with a High Pure FFPET RNA isolation kit (Roche Life Science, Germany (Cat #06650775001)) according to the manufacturer’s instructions. RNA quantification and purity were assessed using a Nanodrop 2000 (Thermo Fisher Scientific). Isolated mRNAs (100 ng sample) were processed through the NanoString nCounter Prep Station (NanoString Technologies, Seattle, WA, USA) on the Hospices Civils de Lyon Platform. Gene expression profiles were established using the human nCounter PanCancer Progression Panel (NanoString Technologies) composed of 770 genes involved in angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis and ECM. Tumor samples were processed according to the manufacturer’s instructions. Normalization of housekeeping genes for the quantification of gene expression levels, normalization of the positive control for background correction and data analysis were performed using the nSolverTM analysis software (NanoString Technologies). Transcripts with an RNA count < 20 for all samples were excluded because they were considered unexpressed. The mean and standard deviation (SD) were calculated for each group. Only genes with significant (p < 0.05) fold changes (< –2 or > +2) were considered as differentially expressed.
4
Nanostring Analysis of Cortical Transcripts
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Total RNA was isolated from both cortical tissue samples and the corresponding cultures at designated time points and processed for Nanostring® nCounter analysis as per manufacturer’s instructions. Briefly, custom probes for the PlexSets were designed by Nanostring to incorporate 18 genes of interest and three housekeeping genes (Supplementary Table 5 ). The probes were ordered from Integrated DNA Technologies and run on a nCounter® PlexSetTM platform and analysed using the nSolverTM Analysis software (Nanostring).
5
Quantitative miRNA Expression Analysis
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For determination of miRNA expression, total RNA was isolated using mirVanaTM miRNA Isolation kit (Life Technologies, USA) as per the manufacturer’s protocol. The RNA quality and concentration was measured using NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, USA). A total of 100 ng of purified total RNA was assayed for determination of 800 human miRNA expression using the nCounter Human v2 miRNA Expression Assay kit (Nanostring Technologies, USA). miRNA expression data normalization was performed using the nSolverTM Analysis Software (Nanostring Technologies) according to the manufacturer’s instructions. In order to avoid using the miRNAs with a very low expression, we further filtered out the miRNAs with expression counts <30 (two times the mean ± 2SD of negative control value, accounting for the background noise). Total 412 miRNAs with expression counts >30 were evaluated for differential expression between sensitive and resistant cell lines and for their correlation with cytarabine chemosensitivity.
6
Transcriptomic Analysis of Pneumococcus-Infected THP-1 Cells
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THP-1 cells were infected with pneumococcus at a multiplicity of infection (MOI) of 10 for 6 h. RNA from infected cells was extracted by RNeasy Mini kit (Qiagen), followed by quantification for gene expression analysis using nCounter® human Immunology v2 panel (NanoString Technologies) through the service provided by Cold Spring Biotech, Taiwan. The generated gene expression data sets were analyzed by nSolverTM analysis software (NanoString Technologies). Background values were corrected from raw data and normalized using 15 housekeeping genes (ABCF1, ALAS1, EEF1G, G6PD, GAPDH, GUSB, HPRT1, OAZ1, POLR1B, POLR2A, PPIA, RPL19, SHDA, TBP, and TUBB). Differential expressed genes (WT/ΔnanA>1 or <1) were further analyzed using the d atabase for a nnotation, v isualization and i ntegrated d iscovery (DAVID, https://david.ncifcrf.gov/ ).
7
Nanostring and Scorecard Analysis of iPSCs
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Nanostring (Nanostring Technologies) and Scorecard analysis was performed as described [17 (link)]. iPSCs were cultured in Freedom-1 medium before RNA isolation. To measure their differentiation propensities, undifferentiated blood-derived iPSC and EBs were collected and total RNA was extracted using RNeasy plus kit (Qiagen). One hundred nanograms of RNA was profiled on the Nanostring nCounter system (Nanostring technologies) according to the manufacturer’s instructions. A custom nCounter codeset for Pluri25 covering 25 genes that evaluate cell pluripotency and a custom nCounter codeset for 3GL covering 83 probes that monitor differentiation were used. Lines were analyzed in biological duplicates for each hESC reference line and iPSC line. All data were normalized analyzed with nSolver Analysis Software (Nanostring Technologies, USA).
8
Profiling Monocyte Transcriptome after IFN-γ
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NanoString-based Gene Expression Profiling
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Gene expression was assessed using the NanoString PanCancer Pathway Panel (NanoString Technologies, Seattle, WA, USA) consisting of probes for 770 genes implicated in carcinogenic pathways, curated from The Cancer Genome Atlas (TCGA) data. All RCC files (direct outputs/raw data from NanoString runs) were normalized using nSolver analysis software (NanoString Technologies, Seattle, WA, USA) according to the manufacturer’s protocols (nSolver User Manual). In brief, a normalization factor was calculated by obtaining the geometric mean of the controls used for each sample and applied to the raw counts of the nCounter output data to eliminate variability that was unrelated to the samples. The resulting data were normalized again with the geometric mean of the housekeeping genes. Normalized data were log2-transformed and exported to Microsoft Excel for analysis.
10
RNA Extraction and NanoString Analysis
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