RNA expression was determined from total RNA isolated using RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). NanoString PanCancer Immuno-Oncology 360TM CodeSet using the nCounter SPRINT platform (NanoString Technologies, Seattle, WA, USA), which includes 750 cancer expression pathway genes, was used to analyze gene expression per manufacturer protocol.
Ncounter sprint platform
Manufactured by NanoString
Sourced in United States
The NCounter Sprint platform is a compact, automated system designed for high-throughput analysis of gene expression. The platform utilizes a proprietary molecular barcoding technology to precisely quantify and profile the expression of multiple target genes simultaneously in a single sample. The NCounter Sprint is capable of measuring gene expression levels across a wide dynamic range and provides a streamlined workflow for sensitive, multiplexed gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Maxwell rsc simplyrna tissue kit, by Promega (2 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Maxwell rsc instrument, by Promega (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nsolver analysis software v4, by NanoString (1 mentions)
High pure ffpet rna isolation kit, by Roche (1 mentions)
Aati fragment analyzer, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
8 protocols using ncounter sprint platform
1
RNA Expression Profiling via NanoString
2
Gene Expression Analysis Using nCounter
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We applied the nCounter in-solution hybridization method using nCounter Sprint platform (NanoString Technologies, Inc. Seattle, WA, USA) to measure the gene expression levels of candidate genes, as previously described [24 (link)]. The cells were treated with GSH or LPS, and incubated for 24 h, and then the total RNAs isolated by collecting the cells. After solution-phase hybridization between the target mRNA and reporter-capture probe pairs, excess probes were removed, and the probe/target complexes were aligned and immobilized in the nCounter cartridge (NCT-120), which was then placed in a digital analyzer for image acquisition and data processing. The raw data was normalized using the housekeeping gene, and the gene expression change was represented by heatmap. The heatmap represented differentially expressed genes by RNA sequencing analysis with fold-change cutoff of 0.5 and 2 (red and green, respectively).
3
Comprehensive Cancer Pathway Analysis
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Total RNA will be extracted from tissue using the using Maxwell RSC simplyRNA Tissue Kit with the Maxwell RSC Instrument (Promega). Prior to gene expression analysis, RNA quality and quantity will be determined using TapeStation4200 (Agilent Technologies) and Qubit Fluorimeter (ThermoFisher). Gene expression profiling will be conducted on the nCounter Sprint platform (NanoString) using the PanCancer Pathways panel, a multiplex gene expression analysis of 770 genes representing 13 canonical pathways in cancer. Barcoded tissue RNA will be analyzed using kits, reagents, and methods per the manufacturer's instructions. NanoString nSolver and Rosalind (Rosalind, Inc.) software will be used to analyze the nCounter‐generated data. P values and fold change values for genes, accounting for background signals in negative controls and normalization factors derived from housekeeping genes and positive controls, will be examined.
4
Gene Expression Profiling with NanoString
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Gene expression on the NanoString nCounter Sprint platform was assessed using total RNA (25–50 ng) with two different panels (PanCancer Progression and PanCancer Pathways; each containing 740 target and 30 “housekeeping” genes) as per the manufacturer’s instructions (NanoString Technologies, Seattle, WA, USA). Table S5 summarizes the samples run, the passage number from which extracts were made, and the panel used.
Data were analyzed using NanoString’s nSolver Analysis Software v4.0 (https://www.nanostring.com ; NanoString Technologies, Seattle, WA, USA) with raw counts normalized using house-keeping genes, selected based on a low coefficient of variation between samples and average counts above negative controls. Differential gene expression was derived using nCounter default settings and quality control governed as per manufacturer’s instructions.
Data were analyzed using NanoString’s nSolver Analysis Software v4.0 (
5
Gene Expression Profiling of Cancer Pathways
6
Immune Profiling of FFPE Tissue Samples
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FFPE tissue blocks were sectioned at a thickness of 3 μm (2-3 sections per block). RNA was extracted using the High Pure FFPET RNA Isolation kit (Roche). The concentration of the extracted RNA samples was measured with Qubit and quality control was performed on a bioanalyzer using RNA6000 pico assay. The percentage of RNA fragments above 200 nucleotides was used to adjust RNA input. Gene expression was interrogated using the PanCancer Immune Profiling and PanCancer Pathways panels (NanoString Technologies) in the nCounter® SPRINT platform. Gene expression data were analyzed using NanoString's software nSolver V.4.0 with Advanced Analysis 2.0 plugin. Data were normalized using the Advanced Analysis tool which draws on the NormqPCR R package.20 (link)
7
Quantifying Gene Expression in Leg Ulcer Biopsies
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Human biopsies from healthy volunteers and patients with venous leg ulcers were collected, as described above, and total RNA was extracted and kept at −80°C. We applied the nCounter in‐solution hybridization method using nCounter Sprint platform (NanoString Technologies, Inc. Seattle, WA) to measure the gene expression levels of candidate genes, as previously described.47
8
Microarray Analysis of Gene Expression
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The nCounter SPRINT platform (NanoString Technologies, Inc. Seattle, WA, USA) was used to analyze the gene expression by microarray, as described previously [21 (link)]. Briefly, RAW 264.7 cells were seeded on 6-well plates at a density of 4 × 105 cells/well, and incubated for 24 h. The cells were treated with CSP32 and l ng/mL LPS for 24 h, and then, the total RNA was isolated by TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. To evaluate for RNA condition, all samples were performed quality control test and quantitative analysis using AATI fragment analyzer (Agilent Technologies, Santa Clara, CA, USA) and DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). After solution-phase hybridization between the target mRNA and reporter-capture probe pairs, the excess probe was removed, and the probe/target complexes were aligned and immobilized in the nCounter cartridge (NCT-120), which was then placed in a digital analyzer for image acquisition and data processing. The raw data were normalized with 14 housekeeping genes, including ALAS1, EEF1G, G6PDX, GAPDH, GUSB, HPRT, OAZ1, POLR1B, POLR2A, PPIA, RPI19, SDHA, TBP, and TUBB5. Fold change data was log2-transformed and log2 fold-change cut-offs of −2.5 and 2.5 (red and green, respectively) in the expression of the selected genes are presented in a heat map.
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