QuantiGene Singleplex AssayGene expression analysis without the limitations

QuantiGene® Singleplex Assay

IntroductionCommon quantitative gene expression methods such as qPCR often present challenges such as enzymatic bias and the need to purify RNA. Furthermore, instrumentation can be costly and researchers often experience difficulty working with certain sample types. By quantitating mRNA or DNA directly from your starting sample and using a signal amplification assay, these challenges can be overcome.

QuantiGene® Singleplex Assay is the most accurate and precise method of quantitating gene expression with a simple workflow.

Key benefits

No bias – avoid the enzymatic bias inherent to reverse transcription and target amplification Faster sample prep – perform the assay directly on cell lysates or tissue

homogenates, eliminating the need for RNA purification and minimizing issues caused by contamination Precisely detect subtle changes – detect changes in gene expression

smaller than 10% rather than the entire fold-changes that are detected by qPCR Works with difficult sample types – quantitate heavily degraded

and cross-linked RNA in FFPE tissues or quantitate RNA in blood, both with ease

Application areas

Screen compounds in drug discovery Validate biomarkers using archival tissue specimens Measure siRNA knockdown efficiency Prospective/retrospective analysis of clinical samples Profile and quantitate miRNAs Microarray validation Predictive toxicology Detect translocations and fusion genes

2

Na2O3PO

CH3

N

S Cl

Samplepreparation

Lyse sampleand go

Chemiluminescentsubstrate

Read signal with a luminometer

Capture extender (CE)

Targethybridization

Signalamplification

Detection

Pre-amplifier

Amplifier

Label probeBlocking probe (BL)

Labelextenders (LE)

Lysate (with target RNA)

Incubate captureplate with sampleand probes

Hybridizations and wash steps

Lysate (with target RNA)

Capture plate Capture probe

Na2O3PO

CH3

N

S Cl Na2O3PO

CH3

N

S Cl

Na2O3PO

CH3

N

S Cl

Samplepreparation

Lyse sampleand go

Chemiluminescentsubstrate

Read signal with a luminometer

Capture extender (CE)

Targethybridization

Signalamplification

Detection

Pre-amplifier

Amplifier

Label probeBlocking probe (BL)

Labelextenders (LE)

Lysate (with target RNA)

Incubate captureplate with sampleand probes

Hybridizations and wash steps

Lysate (with target RNA)

Capture plate Capture probe

Na2O3PO

CH3

N

S Cl Na2O3PO

CH3

N

S Cl

Na2O3PO

CH3

N

S Cl

Samplepreparation

Lyse sampleand go

Chemiluminescentsubstrate

Read signal with a luminometer

Capture extender (CE)

Targethybridization

Signalamplification

Detection

Pre-amplifier

Amplifier

Label probeBlocking probe (BL)

Labelextenders (LE)

Lysate (with target RNA)

Incubate captureplate with sampleand probes

Hybridizations and wash steps

Lysate (with target RNA)

Capture plate Capture probe

Na2O3PO

CH3

N

S Cl Na2O3PO

CH3

N

S Cl

Na2O3PO

CH3

N

S Cl

Samplepreparation

Lyse sampleand go

Chemiluminescentsubstrate

Read signal with a luminometer

Capture extender (CE)

Targethybridization

Signalamplification

Detection

Pre-amplifier

Amplifier

Label probeBlocking probe (BL)

Labelextenders (LE)

Lysate (with target RNA)

Incubate captureplate with sampleand probes

Hybridizations and wash steps

Lysate (with target RNA)

Capture plate Capture probe

Na2O3PO

CH3

N

S Cl Na2O3PO

CH3

N

S Cl

Sample preparation Target hybridization Signal amplification Detection

Technical overview and workflow

QuantiGene® assays utilize clinically proven branched DNA (bDNA) technology that has been used for decades in the VERSANT® 3.0 diagnostic assays for HIV-1, HCV, and HBV. First, cells are lysed or tissue samples are homogenized to release the target RNA. Second, the oligonucleotide probe set is incubated with the target RNA and capture plate overnight. During this incubation, the probes cooperatively hybridize to the target and capture probes immobilized on the plate, capturing the target RNA. Third, signal

amplification is performed via sequential hybridization of the bDNA pre-amplifier, amplifier, and label probe molecules to the target. Addition of a chemiluminescent substrate generates a luminescent signal directly proportionate to the amount of target mRNA present in the sample.

Performance specifications

Limit of detection ≤200 transcripts/well

Limit of quantification ≤500 transcripts/well

Linear dynamic range ≥3.5 logs

Precision (CV)≤10% intra-assay

≤15% inter-assay

Accuracy of fold-change 100% +/- 20%

Specificity ≥99.98%

Plate format 96- or 384-well

3

Exceptional accuracy of measurement – spike recoveries of 85-115%

A wide range of concentrations (0.001-1.0 attomoles) of an IL-6 in vitro transcribed (IVT) RNA was spiked into a cell lysate with undetectable levels of endogenous expression. Spike recovery was calculated as signal from the IVT RNA in the presence of lysate divided by the signal in the absence of lysate multiplied by 100.

% R

eco

very

IL6 IVT RNA (attamole)

0.000 0.001 0.010 0.100 1.000

140

120

100

80

60

siRNA knockdown screening and validation

PMA induction of HeLa cells led to a spike in IL-8, which was then knocked down by siRNA treatment. Knockdown efficiency was measured at two time points and a 7% change was accurately detected by QuantiGene® assays, whereas qPCR can only measure fold-changes.

Rat

io (

IL-8

/ C

yclo

ph

ilin

)

350 fold induction

Δ 7%

87% knockdown

94% knockdown

IL-8 siRNA8 hr post-Rx

IL-8 siRNA4 hr post-Rx

PMA inducedHeLa

Untreaded HeLa

7.000

6.000

5.000

4.000

3.000

2.000

1.000

0.000

Detection of low abundance genes not detected by qPCR

QuantiGene assays were able to detect 3 RNAs of low abundance in two reference samples: brain RNA and universal human reference RNA. These genes were undetectable by qPCR (Ct>35). This exquisite sensitivity allows for the basal measurement of low expression genes.

RLU

C11 orf9 GULP1

10ng/well

Background Brain RNA Universal Human Reference RNA

RECQL4

80

70

60

50

40

30

20

10

0

Quantiative gene expression data from archived FFPE samples

Profiling with QuantiGene® Singleplex Assay of lactate dehydrogenase RNA, in 14-year old FFPE lung tumor (14T) and adjacent normal tissue (14N), demonstrated 2-3 fold induction of this advanced-stage cancer biomarker, in agreement with published data1. RNA purified from these samples was extremely degraded and failed to produce quantifiable signal in qPCR experiments.

1 Beer DG et. al., Gene expression profiles predict survival of patients with lung adenocarcinoma. Nat Med. 2002 Aug; 8(8):816-24.

Fold

Cha

nge

LDHA RPL19 RPL32

3T/3N 14T/14N

5

4

3

2

1

0

In agreement with the literature (Beer, et.al. 2002), QuantiGene® assay detected 2-fold induction of LDHA RNA in tumor relative to normal tissue even in highly-degraded 14 year old samples.

3N 3T 14N 14T

28S

18S

RNA from 3 and 14 year old FFPE samples of tumor (T) and adjacent normal (N) tissue from lung cancer patients as visualized by agarose gel electrophoresis The positions of the 28S and 18S are indicated.

Other QuantiGene® Singleplex Assay highlights

Quick turnaround—thousands of probe designs stocked; custom probes, designed to any sequence, ready to ship within two weeks

Superior specificity—delivers greater specificity than other common technologies, and distinguishes between closely related genes due to probe design with six capture points along the target mRNA of interest

Flexibility—easily automated for use in routine compound screening

Compatibility—works with a variety of sample types: cultured cells, whole blood, PAXgene(R) blood, dried blood spots, fresh or frozen animal or plant tissues, FFPE samples and purified RNA, using standard instrumentation (microplate luminometer and a horizontal airflow oven)

Expanding portfolio—also available for DNA copy number analysis and microRNA expression

Contact your local eBioscience account manager or QuantiGene sales specialist for more information: www.ebioscience.com/contact-us/sales-representatives.htm

Ask us about our QuantiGene® Evaluation Kit, which enables new users to experience the QuantiGene assay’s simple workflow and outstanding precision, accuracy, and robustness.

eBioscience Tel: +1-888-999-1371 Tel: +1-858-642-2058 eBioscience (EU) Tel: +43 1 796 40 40 305 info@ebioscience.com

Affymetrix, Inc. Tel: +1-888-362-2447 Affymetrix UK Ltd. Tel: +44-(0)1628-552550 Affymetrix Japan K.K. Tel: +81-(0)3-6430-4020

Panomics Solutions Tel: +1-877-726-6642 panomics.affymetrix.com USB Products Tel: +1-800-321-9322 usb.affymetrix.com

www.ebioscience.com Please visit our website for international distributor contact information.For Research Use Only. Not for use in diagnostic or therapeutic procedures.

QG04903-1 QuantiGene Singleplex Assay-PLF 0215©2015 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom®, Command Console®, CytoScan®, DMET™, GeneAtlas®, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™, myDesign™, NetAffx®, OncoScan®, Powered by Affymetrix™, PrimeView®, Procarta®, ViewRNA®, and QuantiGene® are trademarks or registered trademarks of Affymetrix, Inc. 123count eBeads™, BestProtocols®, eBioscience®, eFluor®, eVolve™, Full Spectrum Cell Analysis®, InstantOne ELISA™, MagniSort™, OneComp eBeads®, PrimeFlow™, ProcartaPlex™, Ready-SET-Go!®, SAFE™, Super AquaBlue®, The New Standard of Excellence®, and UltraComp eBeads® are trademarks or registered trademarks of eBioscience, Inc. Instant ELISA® is a registered trademark of Bender MedSystems, GmbH. All other trademarks are the property of their respective owners.