TECH NOTE nCounter System Improvements to Fold-Change Sensitivity & Number of Detectable Genes

nCounter

Analysis System

Tech Note

nCounter System Enhancement Provides Improved Fold-Change Sensitivity and Increases the Number of Detectable Genes
Introduction
Since the initial launch of the nCounter Analysis System in 2008 NanoString has maintained an ongoing continuous improvement effort to identify new ways to optimize performance through system enhancements. Our most recent update is focused on optimizing the binding of the puried probe-target tripartite complex to the slide surface, which results in higher and more reproducible digital counts per sample. This optimization involves the priming of the cartridge binding surface prior to sample introduction combined with extending the time provided for binding of the probe-target complex to the slide surface.

Higher and more reproducible counts result in superior data quality with the following benets:
Increased fold-change sensitivity Increased system sensitivity (more genes detectable) Reduced input material option

FIGURE 1: New Prep Plate with green barcode label

Optimization of the binding of the probe-target tripartite complex to the slide surface incorporates two components:
A Prep Station software upgrade which includes two new protocols A new Prep Plate that can easily be identied by its green barcode label (these plates started shipping with all master kits in June 2012)
FIGURE 2: nCounter Prep Station

Two new protocols in the Prep Station software upgrade provide the exibility to both maximize performance and throughput:
High Sensitivity Protocol
Provides maximum fold-change sensitivity Increases the number of detectable genes Improves lane-to-lane reproducibility Enables a 50% reduction of sample input to 50ng (150ng for CNV application) while providing equivalent counts to the current protocol Processing time 3 hours and 5 minutes

Standard Protocol
Improves lane-to-lane reproducibility Processing time 2 hours and 25 minutes

Molecules That Count

Translational Research

Gene Expression

miRNA Expression

Copy Number Variation

Single Cell Expression

TECH NOTE

nCounter System Improvements to Fold-Change Sensitivity & Number of Detectable Genes

The accuracy of fold change measurements at the low end of the dynamic range was examined for the Current and High Sensitivity Prep Station protocols.

signicant fold-change forlow low expressing genes signicant fold change for expressing genes
12
log2 log2 counts counts 2X 2X target target log2 log2 counts counts 2X 2X target target

Current Protocol Protocol Current

signicant fold-change forlow low expressing genes signicant fold change for expressing genes
12 10 8 6 4 2 0 0 2 4 6 1X target 8 log2 counts log2 counts 1X target
Signicantly Different Not Signicant

High Sensitivity Sensitivity Protocol Protocol High

10 8 6 4 2 0 0 2 4 6 1X target 8 log2 counts log2 counts 1X target 10 12

Signicantly Different Not Signicant

10

12

FIGUREs 3A and 3B: A CodeSet containing 143 genes was hybridized to a pool of targets that were present at concentrations ranging from 1.25fM to 0.125fM. The target pool was hybridized at a concentration of 1X and 2X so that each gene should increase 2 fold. Each concentration was run in triplicate and the data was normalized to a set of positive controls that were present at the same concentration in the two target mixes. A t-test was performed between the triplicate measurements at each concentration and the data is shown in FIGURE 3A and 3B. Genes with p-values below 0.05 were considered signicant (green diamonds). Genes that did not change in a statistically signicant manner in response to a 2x increase in target amount are shown in the orange squares. The number of genes that did not signicantly change decreased from 25 genes using the current protocol to only 7 with the High Sensitivity protocol. The number of genes at the lowest concentration (0.125fM) that were not signicant decreased from 16 to 4, a 4 fold reduction. This data demonstrates that the High Sensitivity Prep Protocol improves researchers ability to more accurately measure fold change differences at the low end of the expression scale than was previously possible.

The combination of improved reproducibility combined with increased digital counts enables low expressing genes that were previously below the detection limit of the system to now be detectable.

200 180 160 140 120 100 80 60 40 20 0

GX-Number of Detected Genes

175 136 144 144

120 100 80
ERCC Method No RNA Method

miRNA v2-Number of Detected Genes

104 94

72

60 40 20

62 ERCC Method No RNA Method

Current (100ng)

HS (100ng)

Current (100ng)

HS (100ng)

FIGUREs 4A, 4B and 4C: More genes are detectable using the High Sensitivity protocol. CNV-Number of Detected Genes nCounter assays were each run in triplicate using 100ng of input RNA and processed 500 474 474 474 474 with either the current or the new High Sensitivity Prep Station protocol. In addition, for 450 each assay a set of negative controls were run in triplicate that did not contain RNA. All 400 data was normalized to the ERCC positive controls. Two separate methods were used to calculate the number of detected genes. In the rst method a T-Test was performed 350 on triplicate measurements with the negative ERCC controls (values for all 8 probes 300 ERCC Method were used) against the triplicate measurements for each gene. A gene was considered 250 detected if the average value of the gene was greater than the ERCC negative controls No RNA Method 200 and the p-value was less than or equal to 0.05 (95% condence). The number of genes detected as determined by this method are shown in the green bars (ERCC Method). 150 In the second method, a T-Test was performed on the counts obtained with triplicate 100 measurements for the no RNA lanes vs. those obtained in the presence of 100ng of RNA . 50 The T-Test was performed on a gene-by-by gene basis using the endogenous background 0 values for each gene. For this method, a gene was considered detected if the average Current (300ng) HS (300ng) value of the gene in the 100ng samples was greater than the average value of the no RNA lanes and the p-value was less than or equal to 0.05. The number of genes detected by this method is shown in the gray bars (No RNA Method). The number of genes detected increases when using the High Sensitivity protocol when using either method. Note that for the CNV assay, all genes were detected using either Prep Station protocol. The total number of genes in each assay were 190, 805, and 474 for the GX, miRNA, and CNV assays respectively.

TECH NOTE nCounter System Improvements to Fold-Change Sensitivity & Number of Detectable Genes

The High Sensitivity Protocol provides the equivalent number of counts with a 50% reduction in input material. The bar graphs show comparisons of total counts for increasing amounts of RNA or DNA input using the current and new High Sensitivity Prep Station protocol for the standard nCounter nucleic acid assays (RNA, miRNA and CNV).

mRNA Input Amount

600000 500000 400000 300000 200000 100000 0 Current High Sensitivity 50ng 100ng
250000 200000 150000 100000 50000 0

miRNA Input Amount

50ng 100ng

Current

High Sensitivity

CNV Input Amount

600000 500000 400000 300000 200000 100000 0 Current High Sensitivity 150ng 300ng

FIGUREs 5A, 5B and 5C: Reduced input amounts for High Sensitivity protocol. nCounter assays were each run in triplicate for 50ng and 100ng of input RNA using either the current or the new High Sensitivity Prep Station protocol. Counts for all genes in the CodeSet were summed per lane then averaged for each triplicate. The red horizontal line represents the counts obtained using current recommended input for each assay type (100ng for RNA and miRNA, and 300ng for CNV, gray bar). Half of the recommended input amount is shown in green. For each assay run on the High Sensitivity protocol the amount of counts obtained from half the recommended input amount (i.e., 50ng of RNA and miRNA and 150ng for CNV) generates equivalent or more counts with 50ng when starting with the standard input (green bar is higher than the red horizontal line). Thus, the amount of input material can be reduced by half for each of the nCounter nucleic acid assays with no loss in data relative to the current protocol.

The optimization of the binding of the probe-target tripartite complex to the slide surface has been demonstrated to provide signicant improvements in data quality generated by the nCounter system. Improvements in reproducibility combined with more digital counts results in increased fold-change sensitivity along with improved sensitivity (the ability to detect more low expressing genes). To identify this update on your Prep Station, look for software update Version 4.0.9.1 for GEN1 instruments and 4.0.9.2 for GEN2 instruments. If you have not received instructions regarding how to upgrade your system please contact us at:

1 888 358 6266 or at support@nanostring.com.

NanoString Technologies, Inc.

530 Fairview Ave N Suite 2000 Seattle, Washington 98109

COntact Us
info@nanostring.com Tel: (888) 358-6266 Fax: (206) 378-6288

SaLes COntacts
United States: us.sales@nanostring.com Europe: europe.sales@nanostring.com Japan: japan.sales@nanostring.com Other Regions: info@nanostring.com

www.nanostring.com

2013 NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, nCounter and Molecules That Count are registered trademarks or trademarks of NanoString Technologies, Inc., (NanoString) in the United States and/or other countries. The manufacture, use and or sale of NanoString product(s) may be subject to one or more patents or pending patent applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

v.20130402