Ncounter flex analysis system

Manufactured by NanoString

Sourced in United States

The nCounter FLEX Analysis System is a versatile, automated platform designed for high-throughput gene expression analysis. It utilizes a unique molecular barcoding technology to simultaneously measure the expression levels of hundreds to thousands of genes in a single sample. The system provides a quantitative, reproducible, and scalable solution for a wide range of applications, including gene expression profiling, targeted gene panels, and more.

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24 protocols using ncounter flex analysis system

Circadian Regulation of Metabolism

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The expression of several genes related to glucose metabolism, the circadian system, fasting, autophagy, and oxidative stress was also assessed in the morning and evening. The full set of genes and their accession numbers are listed in Table S1 (Supplementary Materials). Whole blood was collected in Tempus™ Blood RNA tubes, and mRNA was later isolated from frozen samples using Tempus™ Spin RNA Isolation Kit (Applied Biosystems; Foster City, CA, USA). Extracted mRNA was included in the analysis if the concentration exceeded 55 ng/μl and the A260/A280 and A260/A230 ratios were between 2.12–2.26 and 2.07–3.21, respectively, as determined by a DeNovix DS-11 Spectrophotometer (DeNovix, Inc.; Wilmington, DE, USA) reading. These criteria were met at all four time points in eight of the 11 subjects. Samples were analyzed on the NanoString nCounter FLEX Analysis System (NanoString Technologies, Inc.; Seattle, WA, USA), following the manufacturer’s instructions. Data files were imported to the NanoString software nSolver 4.0, normalized using the geometric mean of six selected housekeeping genes (Table S1), and analyzed as fold changes.

Jamshed H., Beyl R.A., Della Manna D.L., Yang E.S., Ravussin E, & Peterson C.M. (2019). Early Time-Restricted Feeding Improves 24-Hour Glucose Levels and Affects Markers of the Circadian Clock, Aging, and Autophagy in Humans. Nutrients, 11(6), 1234.

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RNA Isolation and NanoString Analysis for Organ Transplant

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For expression analysis, RNA was isolated from 15 μm sections using the RNeasy FFPE Kit (Qiagen, Venlo, Netherlands). RNA concentration and purity were measured with a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States), and isolates with a 260/280 nm absorbance ratio below 1.4 were excluded. All samples had a volume of 25 μl H₂O containing 111–393 ng mRNA and were concentrated using a Savant SPD111 SpeedVac (Thermo Fisher Scientific) at 35°C for 24 min to a volume of 2–3 μl. According to the recommendations of manufacturer, after a hybridization and preparation step, gene expression was analyzed with the NanoString nCounter FLEX Analysis System (NanoString Technologies, Seattle, WA, United States) using the nCounter Banff Human Organ Transplant (B-HOT) panel, containing 760 genes and 10 internal reference genes (10 (link)).

Vonbrunn E., Angeloni M., Büttner-Herold M., Müller-Deile J., Heller K., Bleich E., Söllner S., Amann K., Ferrazzi F, & Daniel C. (2022). Can Gene Expression Analysis in Zero-Time Biopsies Predict Kidney Transplant Rejection?. Frontiers in Medicine, 9, 793744.

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RNA Extraction and NanoString Analysis

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Total RNA was extracted from six to twelve sections of FFPE tissue sections using the RNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA yield and purity were assessed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, ME, USA). A 260/280 optical density ratio within 1.7–2.3 and a minimal RNA concentration of 10 ng/µL were required for further processing. The mRNA expression was measured with the NanoString nCounter FLEX Analysis System (NanoString Technologies, Seattle, WA, USA) using 100 ng of total RNA and the pan cancer pathway panel (770 genes). The nCounter CodeSet was hybridized to total RNA for 18 h at 65 °C and nCounter Prep Station loading, and expression quantification with the nCounter Digital Analyzer was performed as recommended by the manufacturer.

Kumbrink J., Bohlmann L., Mamlouk S., Redmer T., Peilstöcker D., Li P., Lorenzen S., Algül H., Kasper S., Hempel D., Kaiser F., Michl M., Bartsch H., Neumann J., Klauschen F., von Bergwelt-Baildon M., Modest D.P., Stahler A., Stintzing S., Jung A., Kirchner T., Schäfer R., Heinemann V, & Holch J.W. (2022). Serial Analysis of Gene Mutations and Gene Expression during First-Line Chemotherapy against Metastatic Colorectal Cancer: Identification of Potentially Actionable Targets within the Multicenter Prospective Biomarker Study REVEAL. Cancers, 14(15), 3631.

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Profiling Immune Gene Expression in FFPE Tumor Samples

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Total RNA was extracted from two to three 5-mm-thick FFPE sections of CT26.WT tumors using the miRNeasy FFPE Kit (Qiagen). RNA integrity and quantity were assessed on the TapeStation 2200 using the RNA ScreenTape System (Agilent). Manufacturer's recommended protocols were followed. RNA was analyzed using the NanoString nCounter FLEX Analysis System and the 770-gene mouse PanCancer Immune Profiling Panel (Nano-String) following the manufacturer's standard XT CodeSet Gene-Expression Assays protocol. Post-hybridization sample processing on the Prep Station using the high-sensitivity setting was followed by data collection on the Digital Analyzer using a scan setting of 555 fields of view. Preprocessing of the raw count data, which included background subtraction of the negative control probes, positive control normalization, and housekeeping gene normalization, was performed in the nSolver 4.0 (NanoString) software using the geometric means and default parameters. All samples included in the analysis fell within the default nSolver QC parameters. The background-subtracted, normalized count data were uploaded to ROSALIND v3.12.0.5 (OnRamp) for differential gene-expression and pathway analyses.

Proia T.A., Singh M., Woessner R., Carnevalli L., Bommakanti G., Magiera L., Srinivasan S., Grosskurth S., Collins M., Womack C., Griffin M., Ye M., Cantin S., Russell D., Xie M., Hughes A., Deng N., Mele D.A., Fawell S., Barry S., Reimer C., Barrett J.C, & McCoon P. (2020). STAT3 Antisense Oligonucleotide Remodels the Suppressive Tumor Microenvironment to Enhance Immune Activation in Combination with Anti-PD-L1. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(23).

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Comprehensive Immune Profiling of FFPE Samples

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RNA extraction and gene expression analysis were performed as previously described. Briefly, three to five consecutive 20-µm sections were obtained from each FFPE block and RNA was isolated using the RNeasy FFPE Kit (Qiagen). RNA concentration and purity were measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Gene expression was quantified using a NanoString nCounter FLEX Analysis System (NanoString Technologies, Seattle, WA), as per manufacturer recommendations. For this study, we utilized the 770-gene nCounter PanCancer Immune Profiling Panel (https://www.nanostring.com/products/gene-expression-panels/gene-expression-panels-overview/hallmarks-cancer-gene-expressionpanel-collection/pancancer-immune-profiling-panel) plus 30 additional custom genes, including five polyomavirus genes (Agnoprotein, LTAg, VP1, VP2, VP3) and 25 additional immune-related genes previously reported to be associated with TCMR. This resulted in a total of 800 genes being analyzed for each sample, including 760 experimental genes and 40 housekeeping genes. Quality control assessment and data normalization were performed using the default settings in nSolver Analysis Software Version 4.0 (NanoString Technologies).

Adam B.A., Kikic Z., Wagner S., Bouatou Y., Gueguen J., Drieux F., Reid G., Du K., Bräsen J.H., D'Agati V.D., Drachenberg C.B., Farkash E.A., Brad Farris A., Geldenhuys L., Loupy A., Nickeleit V., Rabant M., Randhawa P., Regele H, & Mengel M. (2020). Intragraft gene expression in native kidney BK virus nephropathy versus T cell-mediated rejection: Prospects for molecular diagnosis and risk prediction. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(12).

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Profiling Tumor Immune Landscape via NanoString

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Tumours were homogenised with the SpeedMill PLUS (Analytik Jena, Jena, Germany) and RNA was extracted using Phenol:Chloroform:Isoamyl Alcohol (25:24:1) (Sigma–Aldrich, USA) and MagMAX-96 Total RNA Isolation Kit (Thermo Fisher) following manufacturer’s instructions. Extracted RNA was analysed for differential expression by means of the nCounter PanCancer Immune Profiling Panel and the nCounter FLEX Analysis System (NanoString Technologies, Seattle, WA, USA). Profiled data were pre-processed following the manufacturer’s recommendations.^{28 (link),29} Heatmaps of NanoString data were generated using TreeView.^{30 (link)}

Schreiber L.M., Urbiola C., Das K., Spiesschaert B., Kimpel J., Heinemann F., Stierstorfer B., Müller P., Petersson M., Erlmann P., von Laer D, & Wollmann G. (2019). The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer. British Journal of Cancer, 121(8), 647-658.

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Glial Transcriptome Profiling Protocol

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30 to 50 milligrams of frozen DLPFC, primarily gray matter, was homogenized in one milliliter of Trizol with a bullet blender. Chloroform and centrifugation were used to obtain an aqueous phase containing RNA. The aqueous phase was mixed 1:1 in 100% ethanol and then processed with the Quick-RNA Miniprep Kit (Zymo Research, USA). RNA was eluted in nuclease free water and stored at −80 °C until analysis. Quality of the RNA was determined by the Emory Integrated Genomics Core. All samples had concentrations greater than 20 ng/ul and DV₂₀₀ values greater than 20%, per manufacturer’s recommendation. Samples from each disease group were randomly distributed across two nCounter Human glial profiling panels (NanoString, USA) and run on an nCounter FLEX Analysis System (NanoString, USA).

Johnson A.G., Webster J.A, & Hales C.M. (2021). Glial profiling of human tauopathy brain demonstrates enrichment of astrocytic transcripts in tau-related frontotemporal degeneration. Neurobiology of aging, 112, 55-73.

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Profiling Tumor Immune Landscape

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Snap frozen tumor tissue was homogenized using RLT buffer (Qiagen, Hilden, Germany) and SpeedMill PLUS (Analytik Jena, Jena, Germany) followed by Phenol/Chloroform extraction. RNA was isolated from the aqueous phase using RNeasy Mini kit (Qiagen) according to manufacturer’s instructions and total RNA was used for differential expression analysis using the nCounter PanCancer Immune Profiling Panel and the nCounter FLEX Analysis System (NanoString Technologies, Seattle, WA, USA). Profiled data were pre-processed following manufacturer’s recommendations⁵¹ and heatmaps were generated using nSolver 4.0 software. Normalized gene counts from nSolver software were used to calculate the principal component analysis (PCA) using ClustVis^{52 (link)}. Venn diagrams were generated using the webtool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Das K., Belnoue E., Rossi M., Hofer T., Danklmaier S., Nolden T., Schreiber L.M., Angerer K., Kimpel J., Hoegler S., Spiesschaert B., Kenner L., von Laer D., Elbers K., Derouazi M, & Wollmann G. (2021). A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity. Nature Communications, 12, 5195.

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Tumor RNA Profiling for Immuno-Oncology

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Total tumor RNA [tumor cells and tumor microenvironment (TME)] was extracted from four to six 10-μm tumor sections after identification of the residual tumor by a pathologist on a corresponding H&E section. RNA was extracted using the High Pure FFPET RNA Isolation Kit (Roche®, Meylan France), following the manufacturer’s instructions. RNA quantity and quality were assessed by NanoDrop 1000 spectrophotometer (ThermoFisher, Waltham, MA) and 2100 Bioanalyzer with Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA). Two hundred nanograms of total RNA was used from each sample for gene expression profiling (acceptation criteria 260/280 ratio: 1.8-2.3, 230/280 ratio: 1.7-2.3).
RNA samples were analyzed based on the NanoString PanCancer Immuno-Oncology panel (IO360), made of 770 genes related to the interplay between tumor, microenvironment and immune response in cancer. Sample runs were carried out on the nCounter® FLEX Analysis System (automated nCounter® Prep station and the nCounter® Digital Analyzer optical scanner, NanoString Technologies) according to the manufacturer’s protocol. This panel was designed using biological signatures, including the 18-gene Tumor Inflammation Signature (TIS) from Ayers et al.²⁷ (link)

Blaye C., Darbo É., Debled M., Brouste V., Vélasco V., Pinard C., Larmonier N., Pellegrin I., Tarricone A., Arnedos M., Commeny J., Bonnefoi H., Larmonier C, & MacGrogan G. (2022). An immunological signature to predict outcome in patients with triple-negative breast cancer with residual disease after neoadjuvant chemotherapy. ESMO Open, 7(4), 100502.

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NanoString-based Immune Profiling of FFPE Samples

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Tumor cellularity was assessed prior to RNA isolation on HE stained slides and ranged between 60% and 90%. RNA was extracted from three-to-five 10 μm FFPE curls of whole sections using the QIAGEN RNeasy FFPE Kit (Qiagen, Venlo, Denmark) and quantified with Qubit Fluorometer (Invitrogen, Waltham, MA, USA). The RNA samples were diluted to 50 ng/µL.
The NanoString nCounter PanCancer Immune Profiling Panel is a unique 770-plex gene expression panel containing 730 immune-related genes and 40 housekeeping genes (https://www.nanostring.com/products/ncounter-assays-panels/oncology/pancancer-immune-profiling) (accessed on: 14 April 2021) [51 (link)].
According to the manufacturer’s guide, 8 µL of Master Mix (Mixture of Reporter CodeSet and Hybridization Buffer) was added to 5 µL of sample RNA in a tube. After adding 2 µL of Capture ProbeSet to each tube, the solution was gently mixed, briefly spun and placed immediately in a pre-heated 65 °C thermal cycler for 24–26 h. After incubation, the samples were immediately placed into the nCounter Prep station, and then analyzed in the Digital Analyzer (nCounter FLEX Analysis System, NanoString, Seattle, WA, USA). Measurements were taken at high sensitivity with 555 FOV.

Szeitz B., Pipek O., Kulka J., Szundi C., Rusz O., Tőkés T., Szász A.M., Kovács K.A., Pesti A., Ben Arie T.B., Gángó A., Fülöp Z., Drágus E., Vári-Kakas S.A, & Tőkés A.M. (2022). Investigating the Prognostic Relevance of Tumor Immune Microenvironment and Immune Gene Assembly in Breast Carcinoma Subtypes. Cancers, 14(8), 1942.

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