Ncounter sample prep station

Manufactured by NanoString

The NCounter Sample Prep Station is a lab equipment product designed to automate the sample preparation process for the NanoString nCounter Analysis System. It provides a standardized and efficient workflow for preparing samples for gene expression analysis.

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11 protocols using ncounter sample prep station

Profiling Mouse Tumor Immune Responses

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Total RNA was isolated from day 3 fresh mouse tumors after we implanted MC38-fLuc IC in B6 mice (n = 19), harvested solid tumors on day 3 post-implantation. For gene expression analysis, 100 ng total RNA per reaction was used for hybridization using the nCounter PanCancer Mouse Immune Profiling panel, according to the manufacturer’s nCounter XT protocol (NanoString Technologies, Panel XT_PGX_MmV1_CancerImm_CSO XT-CSO-MIP1-12 ref 115000142). The panel include 770 cancer-related mouse genes and 20 internal reference controls. Samples were processed on nCounter Sample Prep Station and nCounter Digital Analyzer (NanoString Technologies). Raw data were extracted using nSolver 2.6 software (NanoString Technologies). Gene expression values were calculated by quantile normalization of log2-transformed data. Groups of mouse tumors were defined using the first principal components analysis (PCA). Differential genes expressions were tested using ANOVA corrected to multiple testing correction by the Benjamin-Hochberg (BH) methods and genes with p-Value <0.05 (Fold Change threshold >1,10; heat map Supplementary Fig. S6a) or <0.005 (Fold Change threshold >1,16; heat map Supplementary Fig. S6b) were selected. Z-score normalized expression of selected genes were illustrated using heat map with unsupervised hierarchical clustering based on euclidean distance.

Trimaglio G., Tilkin-Mariamé A.F., Feliu V., Lauzéral-Vizcaino F., Tosolini M., Valle C., Ayyoub M., Neyrolles O., Vergnolle N., Rombouts Y, & Devaud C. (2020). Colon-specific immune microenvironment regulates cancer progression versus rejection. Oncoimmunology, 9(1), 1790125.

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Probe-based Detection of Lung Gene Fusions

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nCounter Lung Gene Fusion Panel (NanoString Technologies, Seattle, WA, USA) was used for probe-based detection of fusion genes in the selected tumor samples. Hybridization and digital reporter counts were performed following the manufacturer’s instructions for nCounter Vantage Fusion Assays (MAN-10023-09, nCounter XT Assay User Manual). Briefly, 84 ng–300 ng of total FFPE RNA was hybridized to nCounter probe sets for 16 h at 67 °C. Samples were processed using automated nCounter Sample Prep Station (NanoString Technologies). Cartridges were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies) set at 555 fields of view (FoV). Reporter counts were collected using NanoString’s NSolver software version 3.0 and analyzed with fusion threshold set at 50 and imbalance threshold at 15 depending on the background level of absolute raw data count.

Vollbrecht C., Lenze D., Hummel M., Lehmann A., Moebs M., Frost N., Jurmeister P., Schweizer L., Kellner U., Dietel M, & von Laffert M. (2018). RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance. BMC Cancer, 18, 1158.

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Detection of AGK-BRAF Fusion Using NanoString

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Detection of AGK-BRAF fusion transcripts using NanoString nCounter technology (NanoString Technologies, Inc., Seattle, WA) was performed following manufacturer’s instructions. In brief, after incubation of total RNA with nCounter probe sets, samples were processed in an automated nCounter Sample Prep Station (NanoString Technologies, Inc., Seattle, WA). nCounter Cartridges (NanoString Technologies, Inc.) containing immobilized and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.). Reporter counts were collected using NanoString’s nSolver analysis software.

Botton T., Talevich E., Mishra V.K., Zhang T., Shain A.H., Berquet C., Gagnon A., Judson R.L., Ballotti R., Ribas A., Herlyn M., Rocchi S., Brown K.M., Hayward N.K., Yeh I, & Bastian B.C. (2019). Genetic heterogeneity of BRAF fusion kinases in melanoma affects drug responses. Cell reports, 29(3), 573-588.e7.

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Profiling Immune-Related Genes in Cancer

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We studied the expression profiles of 770 immune-related genes using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Inc.). The nCounter assay was performed according to the manufacturer's instructions. The RNA was hybridized with the probe sets for 16 h at 67°C, and the samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc.). Cartridges containing immobilized and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.) that had been set at a data resolution of 555 fields of view. Reporter counts were collected and normalized using the nSolver analysis software (version 3.0; NanoString Technologies, Inc.). The analysis between the two groups was performed using a volcano plot after adjusting for the control group (23 (link)). Sequencing data were deposited into the DNA Data Bank of Japan Sequence Read Archive (accession nos. SAMD00549508-SAMD00549517).

Tsubokura H., Ishida M., Nishigaki A., Yokoe T., Komiya S., Butsuhara Y., Yoshida A., Hisamatsu Y., Hashimoto Y., Tsuzuki-Nakao T., Murata H., Tsuta K, & Okada H. (2022). Significance of placental CD200 expression in patients with preeclampsia: Comparison between early- and late-onset patients. Molecular Medicine Reports, 27(1), 18.

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Immune Gene Expression Profiling in TSQCC

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We performed comprehensive mRNA expression profiling of 770 immune-related genes in 10 patients with TSQCC and two normal controls using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA, USA), according to the manufacturer’s instructions. The RNA was hybridised with the probe sets for 16 h at 67 °C, and the samples were then processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc.). Cartridges containing immobilised and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.) that had been set at a data resolution of 555 fields of view. Reporter counts were determined, log₂-transformed, and normalised using housekeeping genes selected using the nSolver analysis software version 4.0 (NanoString Technologies, Inc.). Fold changes, p values, and adjusted p values were determined using the nSolver analysis software version 4.0 with nCounter Advanced Analysis add-on software version 2.0.115. In particular, adjusted p values were determined using the Benjamini–Yekutieli method to control the false discovery rate^{39 (link)}.

Taniguchi Y., Ishida M., Saito T., Ryota H., Utsumi T., Maru N., Matsui H., Hino H., Tsuta K, & Murakawa T. (2020). Preferentially expressed antigen in melanoma as a novel diagnostic marker differentiating thymic squamous cell carcinoma from thymoma. Scientific Reports, 10, 12286.

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Profiling gene expression using nCounter

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Total RNA was isolated from two to three FFPE tissue sections (10 μm thick) using an miRNeasy FFPE Kit (Qiagen) according to the manufacturer’s instructions. The probe sets were custom designed and synthesized by NanoString Technologies (Seattle, WA), and nCounter assays were performed according to the manufacturer’s protocol. Briefly, 500 ng of total RNA was hybridized to nCounter probe sets for 16 hours at 65°C. Samples were then processed using an automated nCounter Sample Prep Station (NanoString Technologies). Cartridges containing immobilized and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies). Reporter counts were collected using the NanoString’s nSolver analysis software ver. 1, normalized, and analyzed.

Lee M.Y., Ku B.M., Kim H.S., Lee J.Y., Lim S.H., Sun J.M., Lee S.H., Park K., Oh Y.L., Hong M., Jeong H.S., Son Y.I., Baek C.H, & Ahn M.J. (2016). Genetic Alterations and Their Clinical Implications in High-Recurrence Risk Papillary Thyroid Cancer. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 49(4), 906-914.

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Identifying ALK Fusion Partners in CRC and GC

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nCounter anchored multiplex polymerase chain reaction assays were performed according to the manufacturer's protocol (NanoString, Seattle, WA). Briefly, 500 ng of total RNA was hybridized to nCounter probe sets for 16 hours at 65°C. Samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc., Seattle, WA). Cartridges containing immobilized and aligned reporter complex were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.). Reporter counts were collected using NanoString's nSolver analysis software version 1, normalized, and analyzed as previously described [10 (link)]. This technology has been shown to successfully identify novel fusion partners to ALK- and RET-rearranged NSCLC [11 (link), 12 (link)]. To identify fusion partners of ALK in CRC and GC, primers for KIF5B, EML4, KLC1, SMCF1 and C2orf44 were designed and used for analyses.

Lee J., Kim H.C., Hong J.Y., Wang K., Kim S.Y., Jang J., Kim S.T., Park J.O., Lim H.Y., Kang W.K., Park Y.S., Lee J., Lee W.Y., Park Y.A., Huh J.W., Yun S.H., Do I.G., Kim S.H., Balasubramanian S., Stephens P.J., Ross J.S., Li G.G., Hornby Z., Ali S.M., Miller V.A., Kim K.M, & Ou S.H. (2015). Detection of novel and potentially actionable anaplastic lymphoma kinase (ALK) rearrangement in colorectal adenocarcinoma by immunohistochemistry screening. Oncotarget, 6(27), 24320-24332.

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nCounter Gene Expression Profiling

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The nCounter™ gene expression assays were custom-designed and synthesized by NanoString Technologies (Seattle, WA, USA). Hybridization, sample cleanup, and digital reporter counts were performed according to the manufacturer's protocol. RNA was obtained from fresh-frozen tissues using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA concentration was assessed by spectrophotometry using the Nanodrop 8000 (Thermo-Scientific, Wilmington, DE, USA).
Samples were processed according to the gene expression protocol of NanoString Technologies. Briefly, total RNA was hybridized to a multiplexed mixture of custom-designed nCounter™ capture and reporter probes complementary to ALK, ROS1, and RET target sequences (Supplemental Table 2) for at least 16 h at 65°C. The samples were cleaned up and processed using an automated nCounter™ Sample Prep Station (NanoString Technologies). Unhybridized probes were removed, and the hybridization complex was immobilized onto a cartridge and aligned. Fluorescently labeled, color-coded reporters were subsequently imaged on an nCounter™ Digital Analyzer (NanoString Technologies) set at 1155 fields of view. Raw reporter counts were collected using nSolver software v1.0 (NanoString Technologies).

Ha S.Y., Choi S.J., Cho J.H., Choi H.J., Lee J., Jung K., Irwin D., Liu X., Lira M.E., Mao M., Kim H.K., Choi Y.S., Shim Y.M., Park W.Y., Choi Y.L, & Kim J. (2015). Lung cancer in never-smoker Asian females is driven by oncogenic mutations, most often involving EGFR. Oncotarget, 6(7), 5465-5474.

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Custom ALK Panel using NanoString

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The custom ALK panel was carried out using the NanoString nCounter Elements™ protocol per the manufacturer’s instructions. All procedures regarding sample preparation, hybridization, detection and scanning were performed as recommended by NanoString Technologies (NanoString, Seattle, WA). The custom probes (A and B) were designed by IDT (IDT Technologies, Coralville, USA) and contained 35–50 bp each, as previously described by Lira and co-authors [13 (link)]. Probes were diluted to a final concentration of 0.6 nM (probe A) and 3.0 nM (probe B) to create the 30X working probe pools. The total amount of up to 100 ng RNA was used. RNA was hybridized with probe pools, hybridization buffer and TagSet reagents in a total volume of 30 μl and incubated at 67 °C for 20 h. Samples were then loaded to the automated nCounter Sample Prep Station (NanoString Technologies, Seattle, WA), which performed the purification steps and cartridge preparation. Finally, the cartridges containing immobilized and aligned reporter complexes were transferred to a nCounter Digital Analyzer (NanoString Technologies), and expression data were subsequently generated using the high-resolution setting, which takes 600 images per sample.

Evangelista A.F., Zanon M.F., Carloni A.C., de Paula F.E., Morini M.A., Ferreira-Neto M., Soares I.C., Miziara J.E., de Marchi P., Scapulatempo-Neto C, & Reis R.M. (2017). Detection of ALK fusion transcripts in FFPE lung cancer samples by NanoString technology. BMC Pulmonary Medicine, 17, 86.

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nCounter Transcriptome Analysis Protocol

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nCounter assays were performed in duplicate, according to the manufacturer's instructions (NanoString Technologies, Inc, Seattle, WA, USA). Briefly, 500 ng of total RNA was hybridized to nCounter probe sets for 16 h at 65°C. Samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc). Cartridges containing immobilized and aligned reporter complexes were subsequently imaged on the nCounter Digital Analyzer (NanoString Technologies, Inc), set at 1155 fields of view. Reporter counts were collected using the nSolver analysis software version 1 in NanoString, normalized, and analyzed as described below. A detailed description of the assay is given elsewhere36 (link).

Park S., Lee J., Do I.G., Jang J., Rho K., Ahn S., Maruja L., Kim S.J., Kim K.M., Mao M., Oh E., Kim Y.J., Kim J, & Choi Y.L. (2014). Aberrant CDK4 Amplification in Refractory Rhabdomyosarcoma as Identified by Genomic Profiling. Scientific Reports, 4, 3623.

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