Ncounter pancancer pathways panel

Manufactured by NanoString

Sourced in United States

The NCounter® PanCancer Pathways Panel is a laboratory equipment product that enables the simultaneous measurement of the expression of multiple genes associated with cancer pathways. It provides a comprehensive and efficient way to analyze gene expression patterns related to key cancer-associated signaling pathways.

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16 protocols using ncounter pancancer pathways panel

Comprehensive Gene Expression Profiling

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A corrected input of 50 ng of RNA from each sample was hybridized with the nCounter® CodeSet using the nCounter® MAX analysis system. Analysis of the expression profiles for more than 700 genes was performed using the nanoString nCounter® PanCancer Pathways Panel and analyzed with the accompanying nSolver™ 4.0 software as previously reported (nanoString Technologies, Seattle, WA, USA).^{13 ,14}

Monibi F.A., Pannellini T., Croen B., Otero M., Warren R, & Rodeo S.A. (2021). Targeted Transcriptomic Analyses of RNA Isolated from Formalin-Fixed and Paraffin-Embedded Human Menisci. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 40(5), 1104-1112.

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Transcriptome Analysis of NK/T Lymphoma

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According to our IHC grading criteria, 8 PRDM1(+) and 8 PRDM1(−) FFPE samples and 2 samples of normal nasal mucosa were selected from the 58 NK/T lymphoma cases. Total RNA was extracted using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. After determining the RNA quality using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.), 5 PRDM1(+) and 5 PRDM1 (−) specimens (P1, P2, P3, P4, P5 and N1, N2, N3, N4, N5, respectively) that met the criterion of NanoString analysis were identified. The 2 normal nasal mucosa samples were used as blank controls (B1, B2). The NanoString nCounter PanCancer Pathways Panel (NanoString Technologies, Inc., Seattle, WA, USA) includes 770 essential genes representing 13 Canonical Pathways: Notch, Wnt, Hedgehog, TGFβ, MAPK, STAT, P13K, RAS, chromatin modification, transcriptional regulation, DNA damage control, cell cycle (CC), and apoptosis. The NanoString nCounter assay was performed according to the standard protocol of NanoString with analysis and normalization of the raw NanoString data conducted using nSolver Analysis Software v3.0 (NanoString Technologies, Inc.). All procedures associated with mRNA quantification, including sample preparation, hybridization, detection and scanning, were carried out as recommended by NanoString Technologies, Inc.

Liu J., Liang L., Li D., Nong L., Zheng Y., Huang S., Zhang B, & Li T. (2019). JAK3/STAT3 oncogenic pathway and PRDM1 expression stratify clinicopathologic features of extranodal NK/T-cell lymphoma, nasal type. Oncology Reports, 41(6), 3219-3232.

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Validating RNA-Seq with NanoString Profiling

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Gene expression was measured using the NanoString nCounter PanCancer Pathways Panel (NanoString Technologies, Seattle, WA, USA). Each Panel consists of 770 genes, including 13 housekeeping genes. A total of eight tumour samples (four responder and four non-responder) from the same patients as were analysed by RNA-Seq, were subjected to NanoString analysis, to validate RNA-Seq gene expression data, and to perform cancer pathway analysis. The samples were selected based on mapping efficiency (more than 90%) to the human genome (assembly GRCh37) based on our RNA-Seq data analysis. For each NanoString assay, 1 μg of total tissue RNA was isolated, mixed with a NanoString code set mix and incubated at 65°C overnight (16–18 hr). The reaction mixes were loaded on the NanoString nCounter Prep Station for binding and washing, and the resulting cartridge was transferred to the NanoString nCounter digital analyzer for scanning and data collection. Raw count data was preprocessed using the geNorm algorithm in nCounter Advanced Analysis ver. 2.0.115 (NanoString Technologies) (31 (link)). A quality check of raw data was conducted using nSolver Analysis Software ver. 4.0 and NanoStringQCpro ver. 1.14.0 (NanoString Technologies). Z-scores were generated for each gene from normalised data to perform further analysis.

Hossain S.M., Gimenez G., Stockwell P.A., Tsai P., Print C.G., Rys J., Cybulska-Stopa B., Ratajska M., Harazin-Lechowska A., Almomani S., Jackson C., Chatterjee A, & Eccles M.R. (2022). Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures. Frontiers in Immunology, 13, 955063.

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Identifying Cell Signaling Pathways Affected by JQ1

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To identify cell signaling pathways affected by JQ1, multiplex gene expression analysis was performed using the nCounter® PanCancer Pathways Panel. Following JQ1 treatment, RNA was extracted from MDA-MB-231 and MDA-MB-453 cells as described above. The extracted RNA was subjected to an nCounter® PanCancer Pathways Panel (NanoString Technology, WA, USA) according to the manufacturer’s protocol. Briefly, capture probes, reporter probes, and total RNA were mixed with hybridization buffer and hybridized at 65 °C overnight. Then, the samples were processed on the nCounter Prep Station with wash reagents and an imaging cartridge. Hybridization occurred over 20 hours and was monitored in selected experiments.

Park I.H., Yang H.N., Jeon S.Y., Hwang J.A., Kim M.K., Kong S.Y., Shim S.H, & Lee K.S. (2019). Anti-tumor activity of BET inhibitors in androgen-receptor-expressing triple-negative breast cancer. Scientific Reports, 9, 13305.

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NanoString Analysis of Immune and Pathway Genes

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NanoString was performed using the nCounter® PanCancer Immune Profiling panel (XT–CSO-HIP1-12) composed of 770 immune response genes and the nCounter® PanCancer pathways panel (XT–CSO-PATH1-12) comprised of 770 genes from 13 canonical cancer associated pathways (NanoString Technologies). RNA was extracted using the Highpure miRNA isolation kit (Roche) from FFPE blocks, following initial confirmation of tumor presence and content by two pathologists by H&E. For gene expression studies, 1 μg of RNA was used as per manufacturer's instructions (NanoString Technologies). All samples included in this study passed a quality check before the NanoString analysis. Raw NanoString results were normalized using standard NanoString housekeeping genes before comparing tumor-normal pairs. We performed a “Core Analysis” with the significantly differently expressed genes (after Bonferroni correction) in the Cancer Immune and separately with the significant results in the Cancer Pathways panel.

Roszik J., Ring K.L., Wani K.M., Lazar A.J., Yemelyanova A.V., Soliman P.T., Frumovitz M, & Jazaeri A.A. (2018). Gene Expression Analysis Identifies Novel Targets for Cervical Cancer Therapy. Frontiers in Immunology, 9, 2102.

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Comprehensive Transcriptomic Analysis of ER, ECR, and EAR Colonies

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The High Pure RNA isolation Kit (Roche Diagnostics) was employed for RNA extraction from ER, ECR and EAR colonies according to the manufacturer's instructions, and concentrations were estimated using the Qubit 3.0 fluorometer (Invitrogen, Eugene, OR, USA). Purified RNA was analyzed using the nCounter® PanCancer Pathways Panel (NanoString Technologies, Seattle, WA), which includes 730 transcripts and 40 housekeeping genes. The hybridization steps were performed in a Verity thermal cycler (Applied Biosystems, South San Francisco, CA, USA). All processes of capture, cleanup, and digital data acquisition were performed with nCounterPrep StationTM and Digital AnalyzerTM (NanoString Technologies, Seatle, WA, USA) according to the manufacturer's instructions. The number of counts for each gene was extracted from the nCounter generated RCC files using nSolver analysis Software (version 4.0.70 NanoString Technologies) and exported to Microsoft Excel software. RNA counts were normalized using the positive controls and housekeeping transcripts, as described [34] (link). Finally, pathway analysis was performed using nCounter Advanced Analysis 2.0 from nSolver and STRING free software version 11.5 [35] (link). Only pathways identified by both methods in the same colony were considered.

García-Roman S., Garzón-Ibáñez M., Bertrán-Alamillo J., Jordana-Ariza N., Giménez-Capitán A., García-Peláez B., Vives-Usano M., Codony-Servat J., d'Hondt E., Rosell R, & Molina-Vila M.Á. (2024). Vaccine antibodies against a synthetic epidermal growth factor variant enhance the antitumor effects of inhibitors targeting the MAPK/ERK and PI3K/Akt pathways. Translational Oncology, 40, 101878.

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Gene Expression Profiling of FFPE Menisci

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Gene expression profiling of FFPE menisci was conducted using the nCounter® PanCancer Pathways panel as previously reported (NanoString Technologies, Inc.).¹³ A corrected input of 50 ng of total RNA per sample was hybridized with the nCounter CodeSet and analyzed with the accompanying nSolver software. All procedures were performed following the manufacturer’s instructions.

Monibi F.A., Pannellini T., Otero M., Warren R.F, & Rodeo S.A. (2021). Histologic and molecular features in pathologic human menisci from knees with and without osteoarthritis. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 40(2), 504-512.

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Gene Expression Profiling of Human and Mouse Tissues

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A minimum of 100 ng RNA per human tissue sample was analyzed using the nCounter PanCancer pathways panel on the nCounter FLEX Analysis System (NanoString Technologies, Seattle, Washington, USA) at the Institute of Pathology of the LMU in Munich.
A minimum of 30 ng RNA per mouse tissue sample was analyzed using the nCounter Mouse PanCancer pathways panel on the nCounter SPRINT Profiler System (NanoString Technologies) at the Nanostring Core Facility of the University Medical Center Hamburg-Eppendorf in Hamburg.
Nanostring gene expression data was compiled, normalized, and log2-transformed using the nSolver Analysis Software 4.0.70 including the nCounter Advanced Analysis Software 2.0.115.

Dottermusch M., Sumisławski P., Krevet J., Middelkamp M., Voß H., Siebels B., Bartsch H., Sotlar K., Meyer P., Frank S., Korshunov A., Glatzel M., Schüller U, & Neumann J.E. (2021). Co-activation of Sonic hedgehog and Wnt signaling in murine retinal precursor cells drives ocular lesions with features of intraocular medulloepithelioma. Oncogenesis, 10(11), 78.

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Multiplex Gene Expression Analysis of PDXOs

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100 ng of extracted RNA from PDXOs were subjected to multiplex gene expression analyses using nCounter PanCancer Pathways Panel (NanoString Technologies) on the NanoString nCounter SPRINT profiler system as per manufacturer’s instructions. A total of 770 genes (730 cancer-related human genes and 40 internal reference genes) were evaluated. Results were analyzed using nSolver Analysis and nCounter Advanced Analysis software (NanoString Technologies), in which gene expression levels were normalized to the in-built set of positive and negative control genes [44 (link)]. Differential expression of key transcriptomic pathways was compared between the treatment groups (Ixazomib, Dinaciclib, Ixa + Dina) and DMSO control, with fold change and P values calculated based on nSolver’s recommended default settings. A Venn diagram was constructed to determine overlapping genes with a fold change of ≥ ±2 between PDXO1, 11 and 12. Gene set analysis and global significance scores were calculated from differential expression analysis-derived t-statistics for genes in a particular gene set or pathway, as defined by the software.

Lim J.J., Hooi L., Dan Y.Y., Bonney G.K., Zhou L., Chow P.K., Chee C.E., Toh T.B, & Chow E.K. (2022). Rational drug combination design in patient-derived avatars reveals effective inhibition of hepatocellular carcinoma with proteasome and CDK inhibitors. Journal of Experimental & Clinical Cancer Research : CR, 41, 249.

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Multiplex Gene Expression Profiling

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Multiplex gene expression analyses were performed at the Molecular Oncology Research Center-Barretos Cancer Hospital by nCounter® PanCancer Pathways panel (NanoString Technologies™, Seattle, Washington, USA), which allows the evaluation of 770 genes (730 cancer-related human genes, being 124 driver genes and 606 genes from 13 cancer-associated canonical pathways, and 40 as internal reference loci). An average of 100 ng of RNA was used for hybridization. The system analyses for gene expression digital quantification used was the nCounter® SPRINT Profiler (NanoString Technologies™).

Dettogni R.S., Stur E., Laus A.C., da Costa Vieira R.A., Marques M.M., Santana I.V., Pulido J.Z., Ribeiro L.F., de Jesus Parmanhani N., Agostini L.P., dos Reis R.S., de Vargas Wolfgramm dos Santos E., Alves L.N., Garcia F.M., Santos J.A., do Prado Ventorim D., Reis R.M, & Louro I.D. (2020). Potential biomarkers of ductal carcinoma in situ progression. BMC Cancer, 20, 119.

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