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.
Ncounter flex system
Manufactured by NanoString
Sourced in United States
The NanoString nCounter Flex system is a powerful and versatile digital molecular profiling platform. It utilizes color-coded molecular barcodes and digital detection to provide highly sensitive, precise, and multiplexed gene expression analysis. The nCounter Flex system is designed for researchers to accurately quantify and profile gene expression across a wide range of sample types and applications.
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Lab products found in correlation
Ds 11 spectrophotometer, by DeNovix (8 mentions)
Nsolver 4, by NanoString (3 mentions)
Ncounter platform, by NanoString (3 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (3 mentions)
Nsolver 3, by NanoString (2 mentions)
Ncounter mouse pancancer immune profiling panel, by NanoString (2 mentions)
Simpliamp thermal cycler, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Pancancer pathways panel, by NanoString (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Nsolver 2, by NanoString (1 mentions)
Ncounter low rna input amplification kit, by NanoString (1 mentions)
Nsolver analysis software, by NanoString (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Capture probeset, by NanoString (1 mentions)
Reporter codeset, by NanoString (1 mentions)
Qiashredder kit, by Qiagen (1 mentions)
Nsolver 4.0 analysis software, by NanoString (1 mentions)
Ncounter mouse pancancer pathways panel, by NanoString (1 mentions)
Ncounter master kit, by NanoString (1 mentions)
23 protocols using ncounter flex system
1
Profiling Immune Cells in Omentum Tumors
2
NanoString Analysis of Fisetin and Geraldol Effects
3
Omentum Tumor RNA Profiling Using NanoString
4
Omental Tumor Transcriptional Profiling
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At pre‐specified time points, omental tumors were harvested and whole‐tumor mRNA extracted using TRIzol Plus RNA Purification Kit (Life Technologies Corp.). Tumors were mechanically homogenized in 500 uL of TRIzol reagent added and passaged 5‐10 times through an 18 gauge syringe. The remainder of purification was performed as per the manufacturer's instructions. RNA quantification was performed using the DeNovix DS‐11 Spectrophotometer (DeNovix, Inc). Samples were processed on the NanoString nCounter Flex System per manufacturer instructions using the nCounter PanCancer Mouse Immune Profiling panel (NanoString Technologies, Inc), a gene set interrogating 750 cancer‐related genes alongside 20 internal reference controls (full gene list and controls available on manufacturer's website). nSolver 2.6 software (NanoString Technologies, Inc) was used for data analysis. Pathway analysis was performed using Metascape, a web‐based portal for gene list annotation.18 Here, genes with an absolute value fold change greater than or equal to 2 at the week 10 timepoint were collated and entered into the platform, M. musculus selected as input and analysis species, and available express analysis conducted. Fold change represents change in ENT‐treated samples vs vehicle‐treated.
5
Profiling Extracellular Vesicle CircRNAs
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The nCounter Low RNA Input Amplification Kit (NanoString Technologies, Seattle, WA, USA) was used to retrotranscribe and pre-amplify 4 μL of EV-derived RNA in a Verity thermal cycler (Applied Biosystems) following NanoString’s guidelines. Briefly, samples were denatured at 95 °C for 10 min and hybridized for 18 h at 67 °C. Our custom-made nCounter panel (including 78 circRNAs, 6 linear reference genes and 4 mRNAs [30 (link)] was used to analyze EV-derived pre-amplified cDNA according to the manufacturer’s instructions. RCC files containing data outputted by the NanoString nCounter Flex System (NanoString Technologies) from each run were exported to the nSolver Analysis Software (Version 4.0.70, NanoString Technologies).
6
Profiling Mouse Fibrosis Transcriptome
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Whole PEC were isolated as described above and total RNA isolated using Tri-reagent (Thermo Fisher Scientific) as described by the manufacturer. RNA concentration was determined using the Qbit and RNA-BR kit (Thermo Fisher Scientific). Samples were diluted and 100 ng of RNA was processed for running on a NanoString nCounter FLEX system using the nCounter Mouse Fibrosis V2 panel (NanoString Technologies Inc., Seattle, WA). Raw counts were normalized to internal spike-in controls and the expression of 10 stable housekeeping genes using the geNorm algorithm and differential gene expression calculated using the nSolver Analysis software 4.0 Advanced Analysis tool (NanoString Technologies). The datasets for this study can be found in Figshare available under DOI: 10.48420/14635917.
7
Transcriptional Profiling of Sorted Tumor Cells
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Total RNA from sorted CD45− tumor cells was extracted using a QIAshredder kit (QIAGEN) and an RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s protocol. RNA quantification was performed using the DeNovix DS-11 Spectrophotometer (DeNovix, Inc). 100 ng of purified RNA was added to 3 μL of Reporter CodeSet and 2 μL Capture ProbeSet using an nCounter master kit as recommended (NanoString Technologies). Samples were processed on the NanoString nCounter Flex System per manufacturer instructions using the nCounter Mouse PanCancer Immune Profiling panel or the nCounter Mouse PanCancer Pathways panel (NanoString Technologies). Each gene set interrogates 750 cancer-related genes alongside 20 internal reference controls (full gene list and controls available on manufacturer’s website). Differentially expressed genes were identified in nSolver 4.0 Analysis Software (NanoString) as genes with a p value of less than 0.05 versus the respective baseline control. Reactome pathway analysis was performed using the NetworkAnalyst. The NanoString data have been deposited in the NCBI GEO under accession number GSE178135.
8
PanCancer Pathway Gene Expression Analysis
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RNA was analyzed using the NanoString nCounter platform (Seattle, WA, USA) through the UAB NanoString Laboratory (www.uab.edu/medicine/radonc/en/nanostring ). All RNA samples had A260/A280 and A260/A230 ratios in the range 1.8–2.3, as recommended by the manufacturer and determined using a DeNovix DS-11 spectrophotometer (Wilmington, DE, USA). Briefly, 100 ng of each sample was hybridized for 18 h with the reporter and capture probes specific to the human PanCancer Pathways panel, and then processed on the NanoString nCounter Flex system according to the manufacturer’s instructions. This commercially available panel contains 730 genes involved in 13 hallmark cancer pathways (Apoptosis, Cell Cycle, Chromatin Modification, DNA Damage Control, Hedgehog, MAPK, Notch, P13K, RAS, STAT, TGF-β, Transcriptional Regulation, Wnt) as well as 40 housekeeping genes, which serve as internal normalization controls (also see Table S4 ). The samples were read at the standard 280 FOV count; the resultant RCC data files were imported into NanoString nSolver 4.0; and the raw data was used to run through the advanced analysis module. This module selects the best housekeeping genes to use in the analysis through the Gnorm program, and those selected were used to normalize the data.
9
PanCancer Pathway Gene Expression Analysis
10
NanoString PanCancer Pathways Panel Analysis
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Samples were processed for analysis on the NanoString nCounter Flex system using the 770 gene nCounter® PanCancer Pathways Panel (NanoString Technologies, Inc., Seattle, WA, USA), as previously reported (17 (link)). This panel assesses 13 cancer-associated canonical pathways related to basic cancer biology (Notch, Wnt, Hedgehog, Chromatin modification, Transcriptional regulation, DNA damage control, TGF-β, MAPK, STAT, PI3K, RAS, Cell cycle, Apoptosis). Briefly, 100 ng of total RNA, quantified by a Qubit Fluorometric System (Thermo Fisher Scientific, USA), from each sample was hybridized for 21 hours at 65°C, followed by purification and RNA/probe complex immobilization in nCounter PrepStation (NanoString Technologies, Inc., Seattle, WA, USA) and cartridge scanning in a digital analyzer (NanoString Technologies, Inc., Seattle, WA, USA), according to the manufacturer’s protocol. Reading with 280 field-of-views (FOVs) was used in the study samples.
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