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Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

ABSTRACT: BACKGROUND: We assessed NanoString's nCounter Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affinity purification procedure. The second mRNA target specific-sequence and fluorescent-labeled colored coded probe is then used in the detection with the 3-component complex separated on a surface via an applied electric field followed by imaging. We evaluated reproducibility, accuracy, concordance with quantitative RT-PCR, linearity, dynamic range, and the ability of the system to assay different inputs (matched samples of total RNA from Flash Frozen (FF) and Formalin Fixed Paraffin Embedded Tissues (FFPET), and crude tissue lysates (CTL)). FINDINGS: The nCounter Analysis System provided data equivalent to that produced by Taqman(R)-based assays for genes expressed within the ranges of the calibration curves (above ~0.5 mRNA copies per human cell based on an assumption of 10 pg of total RNA per cell). System response was linear over more than two orders of magnitude with typical CVs of ~6% for concentrations above 1 fM (105 molecules per mL). Profiling the industry-standard MAQC data set yielded correlation coefficients of >0.83 for intensity values and >0.99 for measured ratios. Ninety percent of nCounter ratio measurements were within 1.27-1.33 fold changes of the Taqman(R) data (0.34-0.41 in log2 scale) for FF total RNA samples. CONCLUSION: The nCounter Analysis System generated robust data for multi-gene expression signatures across three different sample preparation conditions.

SUBMITTER: Malkov VA

PROVIDER: S-EPMC2688518 | biostudies-other | 2009

REPOSITORIES: biostudies-other

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Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

Malkov Vladislav A VA Serikawa Kyle A KA Balantac Noel N Watters James J Geiss Gary G Mashadi-Hossein Afshin A Fare Thomas T

BMC research notes 20090509

<h4>Background</h4>We assessed NanoString's nCounter Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affi ...[more]

PMID: 19426535

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Project description:Although genetic studies have identified many hundreds of loci associated with human traits and diseases, pinpointing the causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we have enhanced the sensitivity and reproducibility of the massively parallel reporter assay (MPRA), adapting it to identify variants that directly modulate gene expression. We then applied it to over 29,000 single nucleotide and insertion/deletion polymorphisms from 3,965 cis-expression quantitative trait loci (eQTL). We demonstrate strong correlation between our MPRA approach and existing measures of regulatory function, and determine an approximate sensitivity of ~20% with a positive predictive value of 60-65% to detect an eQTL causal allele. We identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. Thus, we have created a resource of concrete leads for understanding the genetic basis of specific phenotypes and illustrate the promise of this kind of approach for comprehensively interrogating how non-coding polymorphism shapes human biology. The study consists of two separate MPRA experiments, a 78,958 oligo (79k study) and a 7,500 oligo library (7.5k). For each library we processed independent transfections into two lymphoblastoid cell lines; in total we completed 5 replicates of NA12878 and 3 replicates of NA19239. For the 79k library we also performed 5 replicate transfections into the hepatocarcinoma cell line HepG2. Raw data is provided as Illumina reads of the 20 bp barcode from the RNA extracted 24 hours post transfection as well as from the plasmid library used for transfection. We also provide oligo/barcode combinations from the âgfp vector in the form of a tab delimited file containing the raw sequence reads of the barcode (column 1) and genomic sequence (column 2). Processed count files are unnormalized counts for each oligo acquired by summing all barcode matches together for each replicate.

Dataset Information

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

Publications

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

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