Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay SystemReport as inadecuate




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BMC Research Notes

, 2:80

First Online: 09 May 2009Received: 03 September 2008Accepted: 09 May 2009DOI: 10.1186-1756-0500-2-80

Cite this article as: Malkov, V.A., Serikawa, K.A., Balantac, N. et al. BMC Res Notes 2009 2: 80. doi:10.1186-1756-0500-2-80

Abstract

BackgroundWe 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.

FindingsThe nCounter™ Analysis System provided data equivalent to that produced by Taqman-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 10 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 data 0.34–0.41 in log2 scale for FF total RNA samples.

ConclusionThe nCounter™ Analysis System generated robust data for multi-gene expression signatures across three different sample preparation conditions.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-2-80 contains supplementary material, which is available to authorized users.

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Author: Vladislav A Malkov - Kyle A Serikawa - Noel Balantac - James Watters - Gary Geiss - Afshin Mashadi-Hossein - Thomas Fare

Source: https://link.springer.com/







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