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 [email protected]
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.