Ncounter pancancer progression panel

Manufactured by NanoString

Sourced in United States, Germany

The NCounter PanCancer Progression Panel is a gene expression profiling tool designed to measure the expression levels of genes associated with cancer progression. It provides a comprehensive analysis of key biological pathways and processes related to tumor evolution and metastasis.

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Lab products found in correlation

Nanodrop, by Thermo Fisher Scientific (4 mentions) Ncounter low rna input kit, by NanoString (3 mentions) Mirneasy kit, by Qiagen (2 mentions) Rna nano chip, by Agilent Technologies (2 mentions) Rneasy plus kit, by Qiagen (2 mentions) Ncounter pancancer progression panel, by Agilent Technologies (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Digital analyzer, by NanoString (1 mentions) Nsolver analysis software version 4, by NanoString (1 mentions) Prism 8.0 for mac, by GraphPad (1 mentions) Ncounter sprint cartridge, by NanoString (1 mentions) Ncounter sprint, by NanoString (1 mentions) Ncounter analysis, by NanoString (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Ncounter digital analyzer, by NanoString (1 mentions)

16 protocols using ncounter pancancer progression panel

EGFR Expression Analysis via NanoString

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EGFR expression in the tumor was analyzed with NanoString analysis. Archival formalin-fixed paraffin-embedded tumor tissue was retrieved and manually macrodissected. Total mRNA was isolated from the macrodissected tumor tissues using a Qiagen miRNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The RNA sample was quantified with NanoDrop (Thermo Scientific, Wilmington, DE, USA) and regarded as adequate if it contained 400 ng at minimum. The sample was subsequently analyzed with the nCounter PanCancer Progression Panel (NanoString, Seattle, WA, USA) according to the manufacturer’s instructions [12 (link)]. NanoString data processing was done with the R statistical programming environment (v3.4.2). Considering the counts obtained for positive control probe sets, raw NanoString counts for each gene were subjected to technical factorial normalization, which was carried out by subtracting the mean counts plus two times the standard deviation from the CodeSet inherent negative controls. Subsequently, biological normalization using the included mRNA reference genes was performed. Additionally, all counts with P > 0.05 after a one-sided t-test versus negative controls plus two times the standard deviation were interpreted as not expressed over basal noise.

Sakai A., Tagami M., Kakehashi A., Katsuyama-Yoshikawa A., Misawa N., Wanibuchi H., Azumi A, & Honda S. (2020). Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment. PLoS ONE, 15(8), e0238120.

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Gene Set Enrichment Analysis of DAPK1 Knockout

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Pre-ranked GSEA was performed using the GSEA V3.0 software^{24 (link),25 (link)} using standard settings and all gene sets included in the Molecular Signatures Database v6.2. As input we used expression fold change values (DAPK1 ko clones vs. HCT116) for the 770 transcripts profiled by the nCounter^® PanCancer Progression Panel (NanoString Technologies Hamburg, Germany). Gene sets were manually categorized into functional gene-set groups representing biological properties relevant to this paper using the following keywords for each category: ECM matrix (keywords: matrisome, ECM, extracellular collagen); EMT/invasion (keywords: mesenchymal, invasive, epithelial cell migration); integrin pathways (keyword: integrin); cell-substrate adhesion (keywords: matrix adhesion, substrate adhesion, focal adhesion); and healing/inflammation (keywords: wound, “inflam”); Cell–cell adhesion (keywords: cell cycle); chromosome organization (keywords: chromosome, chromatin). All gene sets containing the terms “positive regulation of”, “Negative regulation of”, “UP” and “DN” were omitted from the analysis.

Steinmann S., Kunze P., Hampel C., Eckstein M., Bertram Bramsen J., Muenzner J.K., Carlé B., Ndreshkjana B., Kemenes S., Gasparini P., Friedrich O., Andersen C., Geppert C., Wang S., Eyupoglu I., Bäuerle T., Hartmann A, & Schneider-Stock R. (2019). DAPK1 loss triggers tumor invasion in colorectal tumor cells. Cell Death & Disease, 10(12), 895.

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Comprehensive Transcriptomic Profiling of Cancer Progression

Cited 3 times

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The nCounter® PanCancer Progression panel (NanoString Technologies Inc., Seattle WA) is comprised of 740 genes related to cancer progression processes including angiogenesis, extracellular matrix remodeling (ECM), EMT, tumor growth, and metastasis. Details for the probes included in the Progression panel are available on request from NanoString Technologies Inc. using the form at http://www.nanostring.com/support-documents/ncounter-pancancer-progression-panel-gene-list/. We also designed a custom panel of 69 genes related to arylamine exposure and bladder cancer plus 30 housekeeping genes, 8 negative controls, and 6 spike-in hybridization/positive controls overlapping with those in the Progression panel. The custom genes and their annotations are listed in Supplementary Table S2. Genes in the custom panel were selected from in vitro and in vivo arylamine exposure studies [19 (link)–21 (link), 23 (link), 24 (link)] and include 25 genes related to the DNA damage signaling pathway [19 (link), 23 (link)] and 4 genes related to EMT [20 (link), 25 (link)]. We also included several genes related to bladder cancer from The Cancer Genome Atlas (TCGA) [26 (link)–28 (link)], which are highlighted in the Supplementary Table S2.

Kolli R.T., Xu Z., Panduri V, & Taylor J.A. (2021). Differential Gene Expression in Bladder Tumors from Workers Occupationally Exposed to Arylamines. BioMed Research International, 2021, 2624433.

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Profiling Xenograft Tumor Gene Expression

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Gene expression analysis of xenograft tumors (n = 4, each genotype) was performed using the nCounter Pancancer Progression panel (Nanostring Technologies, Seattle, WA) according to the manufacturers’ protocol. RNA was isolated with TRIzol^TM (Invitrogen, #15596026), and 100 ng of total RNA was used for nCounter hybridizations. Data were normalized to endogenous housekeeper controls.

Baechler S.A., Factor V.M., Dalla Rosa I., Ravji A., Becker D., Khiati S., Miller Jenkins L.M., Lang M., Sourbier C., Michaels S.A., Neckers L.M., Zhang H.L., Spinazzola A., Huang S.N., Marquardt J.U, & Pommier Y. (2019). The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis. Nature Communications, 10, 83.

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Quantitative Transcriptional Profiling of BT549 Cells

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RNA was extracted from BT549 cells 72 hours after siRNA transfection using the RNeasy Plus Kit (Qiagen) and quantified using NanoDrop, while quality was assessed using RNA Nano chips (Cat No: 5067–1511) on an Agilent 2100 Bioanalyzer. RNA Samples were then evaluated using the nCounter PanCancer Progression Panel (NanoString) according to the manufacturer’s directions. Alternatively, samples were evaluated using a custom-designed probe panel consisting of 72 test genes and 6 housekeeping genes (see Supplementary Table 1). Briefly, 100 ng of total RNA was hybridized overnight at 65 °C, then run on a NanoString Prep Station at maximum sensitivity. Cartridges were scanned on a NanoString Digital Analyzer at 555 fields of view. Raw count data was normalized using the nSolver analysis software version 3.0, which normalizes samples according to positive and negative control probes and the geometric mean of six housekeeping probes. Genes with normalized counts less than 20 were considered as background and were not included in the analysis. IPA analyses of NanoString data were performed using the Core Analysis function of the IPA software, v.31823283 (Qiagen Bioinformatics).

Ekambaram P., Lee J.Y., Hubel N.E., Hu D., Yerneni S., Campbell P.G., Pollock N., Klei L.R., Concel V.J., Delekta P.C., Chinnaiyan A.M., Tomlins S.A., Rhodes D.R., Priedigkeit N., Lee A.V., Oesterreich S., McAllister-Lucas L.M, & Lucas P.C. (2017). The CARMA3-Bcl10-MALT1 Signalosome Drives NF-κB Activation and Promotes Aggressiveness in Angiotensin II Receptor-positive Breast Cancer.. Cancer research, 78(5), 1225-1240.

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Transcriptional Profiling of PDOs and FFPE

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One hundred ng of total RNA extracted from PDOs and matching formalin-fixed paraffin-embedded (FFPE) biopsies were run with the nCounter PanCancer Progression panel (Nanostring Technologies, Seattle, WA) according to the manufacturer’s instructions. Raw data were normalized using the NanoStringNorm R package version 1.1.21 following recommended parameters and median centered by genes.

Lampis A., Carotenuto P., Vlachogiannis G., Cascione L., Hedayat S., Burke R., Clarke P., Bosma E., Simbolo M., Scarpa A., Yu S., Cole R., Smyth E., Mateos J.F., Begum R., Hezelova B., Eltahir Z., Wotherspoon A., Fotiadis N., Bali M.A., Nepal C., Khan K., Stubbs M., Hahne J.C., Gasparini P., Guzzardo V., Croce C.M., Eccles S., Fassan M., Cunningham D., Andersen J.B., Workman P., Valeri N, & Braconi C. (2018). MIR21 Drives Resistance to Heat Shock Protein 90 Inhibition in Cholangiocarcinoma. Gastroenterology, 154(4), 1066-1079.e5.

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nCounter PanCancer Progression Profiling

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The remaining cDNAs from the AdnaTest were used for RNA profiling by the nCounter PanCancer Progression Panel (NanoString Technologies, WA). The 770 targeted genes in the panel were amplified using the nCounter Low RNA Input Kit (NanoString Technologies) which has been validated and found to be highly efficient and specific [19 (link)]. Briefly, the cDNAs of CTCs were amplified using the multiplex low-input primer pool with 7 cycles of PCR. The PCR-amplified products were then quantified by an Agilent Bioanalyzer 2100 (Agilent Technologies) and hybridized with the nCounter PanCancer Progression Panel following the standard nCounter hybridization protocol.

Dong L., Du X., Lu C., Zhang Z., Huang C.Y., Yang L., Warren S., Kuczler M.D., Reyes D.K., Luo J., Amend S.R., Xue W, & Pienta K.J. (2022). RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients. Materials Today Bio, 17, 100474.

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Profiling Gene Expression Changes

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HTB-4, HTB-5, and HTB-1 cells were treated as described. RNA was extracted and analyzed using the nCounter PanCancer Progression Panel (NanoString Technologies, Inc., Seattle, WA) according to the manufacturer’s protocol (Supplementary Methods). Genes were considered upregulated or downregulated if in cells treated with M7824, M7824mut, or anti-PD-L1 there was a greater than 3-fold change compared to isotype control.

Grenga I., Donahue R.N., Gargulak M.L., Lepone L.M., Roselli M., Bilusic M, & Schlom J. (2017). Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. Urologic oncology, 36(3), 93.e1-93.e11.

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Differential Gene Expression Analysis

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Total RNA was analysed using the 770-gene human nCounter PanCancer Progression panel (NanoString Technologies) according to manufacturer’s instructions. nCounter data were normalised to synthetic positive control targets and to housekeeping genes using nSolver Analysis software (version 4.0) (NanoString Technologies). A minimum mean count threshold was set to 20 counts, yielding a unimodal distribution of binary-logarithm-transformed counts. To remove between-batch variation, we used the pseudoreplicates approach of the removing unwanted variation-III normalisation method^{102 (link)}, applying k = 2. Statistical comparisons between experimental groups were carried out using two-sided Student’s t-tests (for experimental groups with equal variance; F-test) or two-sided Welch’s t-tests (where experimental groups displayed unequal variance; F-test) with Benjamini–Hochberg correction.

Li Mow Chee F., Beernaert B., Griffith B.G., Loftus A.E., Kumar Y., Wills J.C., Lee M., Valli J., Wheeler A.P., Armstrong J.D., Parsons M., Leigh I.M., Proby C.M., von Kriegsheim A., Bickmore W.A., Frame M.C, & Byron A. (2023). Mena regulates nesprin-2 to control actin–nuclear lamina associations, trans-nuclear membrane signalling and gene expression. Nature Communications, 14, 1602.

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NanoString-Based Transcriptome Profiling of MALT1-Silenced BT549 Cells

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RNA was extracted from BT549 cells 72 hours after MALT1 siRNA transfection with RNeasy Plus Kit (Qiagen) and quantitated by NanoDrop. The quality of RNA was analyzed with RNA Nanochips (catalog no: 5067–1511) on an Agilent 2100 Bioanalyzer. RNA samples were then assessed using the nCounter Pan-Cancer Progression Panel (NanoString) according to the manufacturer’s directions. In short, 100 ng of total RNA was hybridized overnight at 65°C, then run on a NanoString Prep Station at maximum sensitivity. Cartridges were scanned on a NanoString Digital Analyzer at 555 fields of view. Raw count data was normalized using the nSolver analysis software version 3.0, which normalizes samples according to positive and negative control probes and the geometric mean of six housekeeping probes. Genes with normalized counts less than 20 were considered as background and were not included in the analysis. Heatmaps were generated by using the UCSC Xena Browser and Morpheus (https://software.broadinstitute.org/Morpheus).
The Core Analysis function within the IPA software (version 31823283; http://www.ingenuity.com) (Qiagen Bioinformatics) was used to perform pathway analyses with the NanoString data. In parallel, GSEA analyses were performed using the GSEA software package (GSEA v2.2.3) and molecular signatures available from the Broad Institute.

Lee J.Y., Ekambaram P., Carleton N.M., Hu D., Klei L.R., Cai Z., Myers M.I., Hubel N.E., Covic L., Agnihotri S., Krappmann D., Bornancin F., Lee A.V., Oesterreich S., McAllister-Lucas L.M, & Lucas P.C. (2022). MALT1 is a Targetable Driver of Epithelial-to-Mesenchymal Transition in Claudin-low, Triple-Negative Breast Cancer. Molecular cancer research : MCR, 20(3), 373-386.

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