Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System

Meghana M. Kulkarni1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.10
DOI:  10.1002/0471142727.mb25b10s94
Online Posting Date:  April, 2011
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Abstract

This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome‐wide (microarrays) and targeted (real‐time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target‐specific, color‐coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35‐ to 50‐base target‐specific sequences. In addition, each reporter probe carries a unique color code at the 5′ end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3′ end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution‐phase hybridization between target mRNA and reporter‐capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color‐coded barcode for that gene is detected, and the barcode counts are then tabulated. Curr. Protoc. Mol. Biol. 94:25B.10.1‐25B.10.17. © 2011 by John Wiley & Sons, Inc.

Keywords: gene expression signature; multiplex analysis; signal transduction; high‐throughput screening; molecular barcode

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Hybridization of Target mRNA to Gene‐Specific Probe Pairs
  • Basic Protocol 2: Data Collection and Analysis
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Hybridization of Target mRNA to Gene‐Specific Probe Pairs

  Materials
  • Sample (see Strategic Planning): total RNA (150 ng per hybridization reaction; −80°C) or cell lysate (from 2,500‐10,000 cells; −80°C)
  • nCounter GX CodeSet (probe pairs for user‐defined target genes; NanoString Technologies; −80°C)
  • Hybridization buffer (room temperature), provided in nCounter Prep Pack
  • RNase‐free, DNase‐free water
  • nCounter Prep Pack (NanoString Technologies), including: racked tips and foil piercers, 12‐tube strips and caps, tip sheaths (for storing tips, to prevent cross‐contamination and unnecessary tip consumption), and cartridge well seals (store at room temperature)
  • Tube‐Strip PicoFuge (Stratagene)
  • Thermocycler or hybridization oven
  • nCounter Prep Station (NanoString Technologies, no. NCT‐PREP‐120)
  • nCounter Sample Cartridges (NanoString Technologies; store at −20°C)
  • nCounter Prep Plates (foil‐sealed 96‐well plates; NanoString Technologies; store at 4°C)
  • Benchtop centrifuge with swinging bucket rotor for plates (e.g., Eppendorf)
NOTE: Sample Cartridges, Prep Plates, and Prep Packs can be purchased together in a comprehensive nCounter Master Kit.

Basic Protocol 2: Data Collection and Analysis

  Materials
  • nCounter Digital Analyzer (NanoString Technologies, no. NCT‐DIGA‐120)
  • nCounter Imaging Oil Applicator and optical oil, provided with Digital Analyzer
  • nCounter Memory Stick (NanoString Technologies), provided with CodeSet
  • Personal computer with Microsoft Excel
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Figures

  •   FigureFigure 25.B1.1 Overview of nCounter Gene Expression Assay. (A) Schematic of target‐specific capture and reporter probes, showing the 5′ reporter barcode and the 3′ biotin capture handle. The orange segment in the capture probe represents a 30‐base sequence common to all capture probes, and the black segment in the reporter probe represents a 60‐base sequence common to all reporter probes. Both are used to remove excess unbound probe. (B) During hybridization, buffer, the CodeSet (capture and reporter probe library) and sample (total RNA or cell lysate) are combined in strip‐tubes and hybridized overnight at 65°C. Hybridization results in the formation of tripartite structures comprising target mRNA bound to specific capture and reporter probes. (C) Strip‐tubes containing tripartite complexes are placed on the Prep Station (left) for automated post‐hybridization processing. The deck layout is shown on the right. (D) The loaded sample cartridge is transferred to the Digital Analyzer (left) for imaging and data processing. A raw image of color‐coded reporter probes bound to complementary target mRNA is shown (middle) along with a schematic of the tabulated counts for target genes (right). Modified with permission from NanoString Technologies.
  •   FigureFigure 25.B1.2 Linearity and reproducibility of the positive spike‐in controls. DNA oligonucleotide targets were spiked into each sample at concentrations of 0.1, 0.5, 1, 5, 10, 50 and 100 fM. No target was added for the negative control probe pairs. (A) Signal (counts) on a log scale vs concentration of the spike on a log scale. Each of three replicate measurements for each spike in baseline Drosophila S2R+ cells (S2R+_1, −2, −3) and insulin‐stimulated S2R+ cells (S2R+_INS40_1, −2, −3) is shown. The replicate measurements overlap completely except at the lowest concentrations. (B) Average signal vs concentration on a linear scale for positive spike‐in controls in both baseline and insulin‐stimulated Drosophila S2R+ samples. The correlation coefficients ( R2) of the linear fit to the average signal are 0.9786 and 0.9803 for baseline and insulin‐stimulated samples, respectively.
  •   FigureFigure 25.B1.3 Reproducibility of the nCounter platform. (A) Scatter plot of normalized background‐subtracted counts for all 100 genes assayed, shown in log scale for technical replicates of baseline S2R+ samples (S2R+_1 vs S2R+_2). Genes were not filtered based on present/absent calls. The R2 value of the linear fit to this data is 0.9999. (B) Scatter plot of normalized background‐subtracted counts for all 100 genes assayed, shown in log scale for technical replicates of insulin‐stimulated S2R+ samples (S2R+_INS40_1 vs S2R+_INS40_2). Genes were not filtered based on detection. The R2 value of a linear fit to this data is 0.9998.

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Internet Resources
  http://www.nanostring.com/
  NanoString Technologies website for detailed information regarding products, related literature, and applications.
  http://www.broadinstitute.org/gsea/msigdb/index.jsp
  The Molecular Signatures Database (MSigDB) is a collection of gene sets, including positional gene sets, curated gene sets, motif gene sets, computational gene sets, and gene ontology gene sets.
  https://www.oncomine.org/resource/login.html
  A database of cancer gene expression profiles.
  http://www.luminexcorp.com
  Supports a bead‐based platform for high‐throughput gene expression signature analysis for the measurement of up to 100 transcripts in many thousands of samples.
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