Ion 540 kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion 540 Kit is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed for use in specific research applications. The kit provides the necessary components and tools for performing the intended functions. The core function of this product is to enable researchers to conduct their work effectively within the intended scope of use.

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Ion 540 Kit-Chef (24 citations) Ion 540 Chip Kit (18 citations) Ion 540 ™ Kit—OT2 User Guide (2 citations) Ion 540 Kit-OT2 (2 citations) Ion 540 kit-chef kit (2 citations)

6 protocols using ion 540 kit

Exosomal miRNA Profiling from Serum

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Exosomal miRNA was extracted from 1 ml of peripheral blood serum using a Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. The quality of miRNA in the eluate was checked using Agilent Small RNA Kit and the concentration was measured with an Agilent RNA 6000 Pico Kit with Bioanalyzer (Agilent Technologies).
Small Library Construction and Ion PGM sequencing were conducted at Thermo Fisher Scientific. Small RNA libraries were prepared with an Ion Total RNA‐Seq kit version 2 (Thermo Fisher Scientific). Then prepared libraries were sequenced with an Ion 540 kit (Thermo Fisher Scientific) on an Ion PGM System.

Miyamoto K., Saiki S., Matsumoto H., Suzuki A., Yamashita Y., Iseki T., Ueno S., Shiina K., Kataura T., Kamagata K., Imamichi Y., Sasazawa Y., Fujimaki M., Akamatsu W, & Hattori N. (2022). Systemic Metabolic Alteration Dependent on the Thyroid‐Liver Axis in Early PD. Annals of Neurology, 93(2), 303-316.

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Ion Torrent Tumor Mutation Load Assay

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20 ng DNA quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) was used for library preparation with the Oncomine Tumor Mutation Load Assay (Thermo Fisher Scientific) according to manufacturer’s instructions. Library concentrations were quantified with the Ion Library TaqMan Quantification Kit (Thermo Fisher Scientific). Libraries were loaded on the Ion Chef for template preparation and chip loading using the Ion 540 Kit (Thermo Fisher Scientific), followed by sequencing on the Ion S5 XL System (Thermo Fisher Scientic).
Quality of the Ion S5 XL run was assessed with the Ion Torrent Suite 5.10 (Thermo Fisher Scientific). Data were analyzed with the Ion Reporter 5.10 (Thermo Fisher Scientific).

Heydt C., Rehker J., Pappesch R., Buhl T., Ball M., Siebolts U., Haak A., Lohneis P., Büttner R., Hillmer A.M, & Merkelbach-Bruse S. (2020). Analysis of tumor mutational burden: correlation of five large gene panels with whole exome sequencing. Scientific Reports, 10, 11387.

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Targeted NGS for Cell-Free DNA Analysis

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For NGS, a library was prepared using the Oncomine Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific) targeting 52 cancer-associated genes (Additional file 1: Table S1). The NGS panel is designed to detect single nucleotide variations, small indels, copy-number alterations, and gene fusions. The libraries were prepared using > 5 ng nucleic acid input following the Oncomine Pan-Cancer Cell-Free Assay user guide. Templating and sequencing were performed using the Ion 540™ Kit on the Ion Chef™ and on the Ion S5 XL system (Thermo Fisher Scientific). Alignment to the hg19 human reference genome and variant calling were performed using the Torrent Suite™ and Ion Reporter™ software version 5.10, respectively. The Torrent Suite™ Software provided molecular coverage depth and read coverage depth at the target base; therefore, this software was able to increase the detection sensitivity for low-frequency variants [16 (link), 17 (link)].
The average of median read coverage and median molecular coverage were 36,095 × and 1934 ×, respectively. Variants with an allele frequency of > 0.1% were reported. The measured allele frequency (%) was calculated as the mutant coverage depth divided by the total coverage depth.

Kim Y., Shin S, & Lee K.A. (2021). Exosome-based detection of EGFR T790M in plasma and pleural fluid of prospectively enrolled non-small cell lung cancer patients after first-line tyrosine kinase inhibitor therapy. Cancer Cell International, 21, 50.

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Transcriptome Analysis of MSCs under Varying Cell Density

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For transcriptome analysis, RNA was prepared from the MSC obtained under low MNC density (1.25 × 10⁵ cells/cm²) and high MNC density (1.25 × 10⁶ cells/cm²). The total RNA was isolated from cells using Maxwell RSC simplyRNA Cell Kit (Promega, Madison, WI, USA). Ion Ampliseq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for library creation. An Ion Proton next-generation sequencer library of analysis beads was created, and an Ion 540 Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for sequencing using an Ion Proton next-generation sequencer. Gene set enrichment analysis (GSEA) were integrated by R programming software. Protein-coding differentially expressed genes (DEGs) in MSCs obtained by lower MNC density (1.25 × 10⁵ cells/cm²) were identified based on a fold change cutoff of 2.0 in comparison with MSCs obtained by high MNC density (1.25 × 10⁶ cells/cm²).

Nagai H., Miwa A., Yoneda K., Fujisawa K, & Takami T. (2023). Optimizing the Seeding Density of Human Mononuclear Cells to Improve the Purity of Highly Proliferative Mesenchymal Stem Cells. Bioengineering, 10(1), 102.

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Comprehensive Liquid Biopsy Profiling of Breast Cancer

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The ctDNA samples were amplified using the Oncomine Breast cfDNA Assay v2, which covers single nucleotide variations and mutations in AKT1, EGFR, ERBB3, ESR1, KRAS, PIK3CA, TP53, FBXW7, SF3B1 and copy number alterations in CCND1, ERBB2, FGFR1. The resulting libraries were quantified using the Ion Library TaqMan^® Quantitation Kit (Thermo Fisher, Waltham, MA, USA). The prepared libraries were then sequenced on an Ion S5 XL Sequencer using the Ion 530^™ kit and Ion 540^™ kit (Thermo Fisher, Waltham, MA, USA). Somatic variants were identified using Sanger sequencing for allele mutation frequencies ≥30%. The Catalogs of Somatic Mutations in Cancer (COSMIC), ClinVar, and dbSNP were used to identify somatic variants. Data analysis was done via Oncomine TagSeq Breast v2 Liquid Biopsy 2.0 default options with minimum molecular cutoff of 2 and minimum mutant allele frequency of 0.05% (minimum variant molecular count – 0.5/molecular coverage). Whole exome sequencing (WES) was performed on matched tumor DNA (n=20).

Shim H., Kwon M.J., Park I.H., Kim M.K., Cho E.H., Lee J., Lee S.T., Sim S.H., Lee K.S., Kim Y.H., Kim S.K., Lee E.S, & Kong S.Y. (2022). Targeted next generation sequencing of circulating tumor DNA provides prognostic information for management in breast cancer patients. Annals of Translational Medicine, 10(2), 28.

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Oncomine™ Pan-Cancer Cell-Free Assay for cfDNA Sequencing

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A total of 30 samples (19 LB samples and 11 FFPE samples) derived from 14 patients were sequenced. Oncomine™ Pan-Cancer Cell-Free Assay (off-the-shelf panel that targets 272 amplicons within 52 known cancer genes) was used for library preparation using 2.5 to 20 ng of the tumor or cfDNA. The libraries were synthesized manually following the manufacturer’s protocol, quantified with an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA), diluted to 100 pM, and pooled for automated templating with an Ion 540™ kit for the IonChef Instrument. Sequencing was performed with the GeneStudio S5 system and Ion 540™ chips (4–6 LB samples/chip and 10 FFPE samples/chip). The average total mapped reads per sample was 5.2 mln for FFPE samples and 14.2 mln for LB samples with an average coverage of 15,298 for FFPE samples and 44,115 for LB samples. The median molecular coverage was 2842 for LB samples and 921 for FFPE samples.

Olbryt M., Rajczykowski M., Bal W., Fiszer-Kierzkowska A., Cortez A.J., Mazur M., Suwiński R, & Widłak W. (2021). NGS Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Melanoma Samples Using Oncomine™ Pan-Cancer Cell-Free Assay. Genes, 12(7), 1080.

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