Gene expression of inducible breast cancer cell lines BT549, CAMA-1, HCC1428, Hs578T, MCF7, T47D, and ZR-75-1 was assessed using the NanoString platform (NanoString Technologies, Seattle, WA, USA), which has been described previously [28] (link). Briefly, sample RNA was hybridized with a custom 345-gene codeset (NanoString Technologies, WA, USA) at 65°C for 16 hours. Hybridized probe:target mixture was then purified and quantified via nCounter MAX Digital Analyzer (NanoString Technologies, WA, USA). The custom 345-gene codeset used has been described previously [29] (link) and includes ABCB1 as a profiling target (Suppl. Table 3). Raw counts were normalized to internal positive control probes and housekeeping genes according to default parameters in nSolver version 4.0 (NanoString Technologies, WA, USA), with background threshold set to 20. Normalized counts data are available at https://github.com/jasminerethmeyer/ABCB1 .
Ncounter max digital analyzer
Manufactured by NanoString
Sourced in United States
The NCounter MAX Digital Analyzer is a laboratory instrument designed for high-throughput gene expression analysis. It uses a digital technology to count and quantify individual molecules of targeted RNA, DNA, or protein sequences in a single experiment. The instrument provides a sensitive and precise method for measuring gene expression levels in a wide range of biological samples.
Automatically generated - may contain errors
3 protocols using ncounter max digital analyzer
1
Breast Cancer Cell Line Gene Expression
2
NanoString nCounter Gene Expression Profiling
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The NanoString nCounter platform gene expression assay has been described previously [20 (link)]. Briefly, the NanoString nCounter platform assays gene expression directly from RNA samples via hybridization of samples with a set of multiplexed nucleotide probes. Probes for each gene target are uniquely barcoded with a series of fluorophores. Fluorescence microscopy imaging of sample-hybridized fluorophore-labeled probes generates quantitative counts data for each gene in each sample.
For gene expression profiling on the nCounter system, patient sample or HMEC control RNA was first hybridized with the custom 345-gene codeset (NanoString Technologies, WA, USA) at 65 °C for 16 h. Post-hybridization probe:target mixture was then purified and quantified via nCounter MAX Digital Analyzer (NanoString Technologies, WA, USA).
For gene expression profiling on the nCounter system, patient sample or HMEC control RNA was first hybridized with the custom 345-gene codeset (NanoString Technologies, WA, USA) at 65 °C for 16 h. Post-hybridization probe:target mixture was then purified and quantified via nCounter MAX Digital Analyzer (NanoString Technologies, WA, USA).
3
Quantification of Biomarker Transcripts Using NanoString
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The hybridized sample was prepared on chip using nCounter MAX Prep Station and GPS transcripts were quantified using a NanoString Digital Analyzer (NanoString Technologies, Seattle, WA, USA). A total of 100 ng of RNA was used as sample input in a hybridization assay (NanoString Technologies, Seattle, WA, USA). Each hybridized sample was prepared on a cartridge using nCounter MAX Prep Station and an individual transcript of a biomarker signature was quantified using nCounter MAX Digital Analyzer (NanoString Technologies, Seattle, WA, USA). The control criteria for the NanoString analysis were imaging quality: >0.75; linearity: >0.95; limit of detection (LOD): (POS_E/LOD) >1 and binding density of 0.05 < BD < 2.25.
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