12
Quantitate up to 80 genes from cell lysates or tissue homogenates QuantiGene assays for analysis of gene expression or copy number variation
Quantitate up to 80 genes from cell lysates or tissue homogenates
QuantiGene assays for analysis of gene expression or copy number variation
2
QuantiGene Singleplex AssaysAccurate and precise RNA quantitation in a simple workflow
QuantiGene Singleplex AssaysBy quantitating mRNA or DNA directly from your starting sample and using a signal amplification assay, Invitrogen™ QuantiGene™ Singleplex Assays are an accurate and precise method of quantitating gene expression with a simple workflow.
Technical overview and workflowQuantiGene assays utilize 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 bound to the plate, capturing the target RNA. Third, signal amplification is performed via sequential hybridization of the bDNA preamplifier, amplifier, and label-probe molecules to the target. Addition of a chemiluminescent substrate generates a luminescence signal directly proportional to the amount of target mRNA present in the sample.
Key benefits• Fast sample prep—perform the assay directly on cell lysates or tissue
homogenates, no need for RNA purification
• Precisely detect subtle changes—detect gene expression changes smaller than 10%
• Works with difficult sample types—easily quantitate heavily degraded and cross-linked RNA in formalin-fixed, paraffin-embedded (FFPE) tissues, or quantitate RNA in blood
• Quick turnaround—thousands of probe designs stocked; custom probes designed to any sequence, and ready to ship within a few days
• Superior specificity—delivers greater specificity than other common technologies, and distinguishes between closely related genes due to probe design with several 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™ 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)
Na2O3 PO S Cl
Chemiluminescentsubstrate
Na2O3PO S Cl S ClNa2O3PO
Lyse sample
Read signal witha luminometer
Hybridizations and wash steps
Lysate (with target RNA)
Samplepreparation
Targethybridization
Signalamplification
Detection
Capture extender (CE)
Blocking probe (BL)
Labelextenders (LE)
Lysate (with target RNA)
Incubate captureplate with sampleand probes
Capture plate Capture probe
Preamplifier
Amplifier
Label probe CH3
N
CH3
N
CH3
N
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.Percent recovery of the spike was calculated based on the signal from the IVT RNA in the presence of lysate.
Re
cove
ry (%
)
IL6 IVT RNA (attomole)
0.000 0.001 0.010 0.100 1.000
140
120
100
80
60
siRNA knockdown screening and verificationPhorbol myristate acetate (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.
Rat
io (
IL-8
:cyc
lop
hili
n)R
ela
tive
lum
ine
sce
nc
e u
nit
(R
LU
)
350-fold induction
Δ7%
87% knockdown 94% knockdown
IL-8 siRNA8 hr post-treatment
IL-8 siRNA4 hr post-treatment
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 QuantiGene assays were able to detect RNAs from 3 low-abundance genes in two reference samples: brain RNA and universal human reference RNA. These genes were undetectable using other technologies. This exquisite sensitivity allows for the basal measurement of low-expression genes.
C11 orf9 GULP1
10 ng/well
Background
Brain RNA
Universal Human Reference RNA
RECQL4
80
70
60
50
40
30
20
10
0
Quantitative gene expression data from archived FFPE samplesProfiling with a QuantiGene Singleplex Assay for lactate dehydrogenase RNA in 14-year old FFPE lung tumor (14T) and adjacent normal tissue (14N) demonstrated a 2–3 fold induction of this advanced-stage cancer biomarker, in agreement with published data [1].
Fold
cha
nge
LDHA RPL19 RPL32
3T/3N
14T/14N
5
4
3
2
1
0
In agreement with the literature [1], QuantiGene assay detected a 2-fold induction of LDHA RNA in tumor relative to the 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 RNAs are indicated.
Applications include compound screening of drug candidates, biomarker verification, siRNA knockdown efficiency, prospective and/or retrospective analysis of clinical samples, miRNA profiling, microarray verification, predictive toxicology, and detection of translocations and fusion genes.
4
QuantiGene Plex AssaysAccurate and precise RNA quantitation of up to 80 genes in a single well
Overview of QuantiGene Plex AssaysThe Invitrogen™ QuantiGene™ Plex Assays provide an accurate and precise method for multiplexed gene expression quantitation. Using Luminex® xMAP® technology, QuantiGene Plex Assays allow for simultaneous measurement of 3–80 mRNA species in every well of a 96-well plate. QuantiGene Plex Assays also incorporate the exclusively licensed branched DNA technology from Siemens. Branched DNA assays allow for the direct measurement of RNA transcripts by using signal amplification rather than template amplification.
The assays are simple and easy to use; QuantiGene Plex Assays don’t require RNA extraction, cDNA synthesis, or PCR amplification. QuantiGene Plex Assays are ideal for verifying Applied Biosystems™ GeneChip™ analysis or next-generation sequencing (NGS) data and for verifying biomarkers for translational research.
Key features and benefits of the assay• True multiplexing—measure up to 80 genes of interest and housekeeping
genes in the same well with no cross-reactivity
• Standardized platform—96-well plate format compatible with Luminex® 100/200™, MAGPIX®, and FLEXMAP 3D® systems
• Simple workflow—ELISA-like workflow for direct hybridization of transcripts to beads and transcript labeling
• Works with difficult sample types—works with degraded and crosslinked RNA in FFPE tissues, and directly with blood
• Large inventory of verified genes—over 15,000 genes can be mixed to create pathway- and disease-themed panels
• Fast customization—if we don’t have your gene(s), we can create and QC your custom panel within 2 weeks
• ISH compatible—same technology utilized in the Invitrogen™ QuantiGene™ ViewRNA™ Kits for mRNA in situ hybridization (ISH)
Lyse sample
Luminex beadswith capture probes
Streptavidin phycoerythrin (SAPE)
Read signal using a Luminex instrument
Capture extenders (CE)
Incubate beads with sample and probes
Preamplifier
Amplifier
Label probe
Label extenders (LE)
Blocking probes (BL)
Lysate (with target RNA)
Hybridizations and wash steps
Lysate (with target RNA)
80 RNA targets per well
Samplepreparation
Targethybridization
Signalamplification
Detection
5
Sample/gene Mouse gene A Human gene 1 Mouse gene B Human gene 2 Mouse gene C Human gene 3
Background (no RNA) 3 3 7 8 7 7
Human liver RNA 4 24,788 9 24,882 3 1,084
Mouse spleen 3,989 19 7,655 1 18,023 4
Mouse liver 392 4 1,488 2 26,586 2
Xenograft spleen 3,321 27,642 472 24,605 24,563 6,547
Xenograft liver 2,959 21,790 2,114 7,058 27,826 1,702
Application 1: Patient-derived xenograft (PDX) modelA 10-plex QuantiGene Plex panel was developed to simultaneously measure the expression of human and mouse genes in a PDX model. The panel comprised 3 human genes of interest and 2 human housekeeping genes, as well as 3 mouse genes of interest and 2 mouse housekeeping genes. Patient-derived tumors were implanted into JAX™ NOD scid gamma (NSG) mice. After growth and metastasis of the tumor in mice, both the liver and spleen from the mice were harvested, and lysates from untreated and xenografted mice were tested alongside human liver RNA. The table below shows the
QuantiGene Plex Assay applicationsQuantiGene Plex Assays for mRNA profiling and DNA copy number determination are ideal for supporting drug discovery and development efforts, as well as translational and clinical research. The examples that follow highlight the capabilities and benefits of this powerful research tool.
mean fluorescence intensity values for the 6 genes of interest that were obtained with a Luminex® instrument. The data demonstrated assay specificity: human samples were negative for mouse transcripts while untreated mice samples were negative for human transcripts. As expected, tissues from xenografted mice contained both human and murine transcripts. Benefit: The specificity of each component assay within a customized multiplexed QuantiGene Plex panel allows the assay to be tailored to address complex biological questions.
Target discoveryTarget identification and
verification of cell-based microarrays
Lead optimizationHigh-throughput screening
Secondary screening
Preclinical studiesADME
Toxicology
Clinical studiesBiomarker verification
Clinical trials (FFPE tissues)
Basal Luminal
HC
C70
HC
C31
53M
DA
MB
468
SU
M13
14B
T20
H55
78T
BT5
49M
DA
MB
157
MD
AM
B23
1M
DA
M45
3LY
2S
UM
185P
ET4
7DH
CC
202
BT4
74ZR
751
MD
AM
B36
1A
U56
5S
KB
R3
EN1FOXC1GABRPERBB2CA12AGR2TFF3GATA3ESR1SCNN1
101
100
10−1
10−2
10−3
10−4
100
10–¹
10–²
10–³
10–4
10–5
p40 p35 TNF p19 EB13 p28
p40 p35 TNF p19 EB13 p28
qPCRassay
QuantiGenePlex Assay
LPS+ LPS
Flu
ore
scen
ce/µ
g R
NA
2– ∆∆
CT
Color key
Row z-score
SCC
AC
p63+
p63–
−3 −2 −1 0 1 2 3
S100A8KRT5CTA-55I10.1TRIM29S100A2FAT2CLCA2DSG3DSC3TP63CSTADENND2CUSH1CTSPAN8KIF12HGDAC105391.3PROM1DDAH1SPINK1EPS8L3RGL3IL20RBSLC44A4TSPAN12TFF3
V554
V553
V558
V611
B
V562
V568
V572
V636
V629
V509
V622
V615
V524
V669
-2
V548
V545
V505
Histology
p63
SKBR3HeLa
HER2 = 2 HER2 = 14
0
1
2
3
4
5
6
7
8
9
MAPKAPKCh 1
MYSTCh 5
BRFCh 8
FGFRCh 8
ERBBCh 17
GRBCh 17
PNMTCh 17
SMARCE1Ch 17
No
rmal
ized
to
ski
n �b
rob
last Normal skin fibroblast
MDA-231
SKBR3ERBB2 (HER2)/normal
Ratio = 6.5
Clofibrate
0
10
20
30
Fo
ld in
du
ctio
n vs
. veh
icle
co
ntro
l
Fold induction of mRNA
Rifampin
3-MethylcholanthrenePhenobarbitalRitonavirOmeprazolePhenytoinß-Napthoflavone
mRNA CYP panel
1A1 1A2 1E1 2A6 2C6 2C9 2C18 2C19 2B6 2D6 2E1 3A4 3A5
6mm
6 mm6 mm
Liver FFPE sectionsSection 1
Section 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Mea
n �u
ore
scen
ce in
tens
ity
Genes
FFPE liver sections—1 vs. 2
0
400
800
1,200
1,600
6 mm
H&E-stainedsection 1
Unstainedsection 2
–5 1050
–4
–2
0
2
4
pca2$x[.1]
pca
2$x[
.2]
Gene P value
BCL6 1.28 x 10–9
PTEN 1.03 x 10–8
ARPC2 2.17 x 10–8
CXCL12 1.37 x 10–7
BRAF 4.18 x 10–7
PCNA 6.33 x 10–7
CLEC3B 1.13 x 10–6
Metastatic melanomaNormal PCA
Principal component analysis of 7 genes
M17
M17: Not melanoma; lymph node with pigmented histiocytes
6
0
1
2
3
4
5
7
CD86 IL2RACCL8IL8CXCL5 Cig5CXCL10TNFalP6IFNGIL1R1 IFIT3
Rat
io o
f CXCL10
-tre
ated
to
unt
reat
ed
CXCL10 stimulation of whole blood for 6 hr and QuantiGene analysis of gene expression
0.3 µg CXCL10
1.0 µg CXCL10
1.0 µg CXCL10 Heat-inactivated
Patient-derived xenograft(PDX) models
Tumor cell line characterization for drug compound screening
Verification of genes involved with inflammation
FFPE analysis of genes involved with cancer
DNA copy number for breakpoint analysis
CYP genes used with human hepatocytes
Verification of genes in FFPE samples
PCA analysis of genes involved with melanoma
Expression analysis from whole human blood
6
Basal Luminal
HC
C70
HC
C31
53M
DA
MB
468
SU
M13
14B
T20
H55
78T
BT5
49M
DA
MB
157
MD
AM
B23
1M
DA
M45
3LY
2S
UM
185P
ET4
7DH
CC
202
BT4
74ZR
751
MD
AM
B36
1A
U56
5S
KB
R3
EN1FOXC1GABRPERBB2CA12AGR2TFF3GATA3ESR1SCNN1
Application 2: Cell line characterization for compound screeningBreast cancer cell lines were created from primary tumors that contained recurrent genetic abnormalities. A QuantiGene 12-plex assay was developed to differentiate patterns of RNA expression across the cell lines. When the normalized levels of gene expression were plotted on a heat map, the data showed that the pattern of expression for the 12 genes could be used to classify the cell lines as being either the basal or the luminal subtype of breast cancer. The cell lines were then used for further screening of drug compounds based on the specific subtypes. Data courtesy of Joe Gray and Nick Wang, Oregon Health Sciences University.
Benefit: Multiplexing allows for rapid classification of cell lines that can then be used for compound screening.
101
100
10−1
10−2
10−3
10−4
100
10–¹
10–²
10–³
10–4
10–5
p40 p35 TNF p19 EB13 p28
p40 p35 TNF p19 EB13 p28
qPCRassay
QuantiGenePlex Assay
LPS+ LPS
Flu
ore
scen
ce/µ
g R
NA
2– ∆∆
CT
Application 3: Target verificationGene expression signatures for inflammation pathway activation were identified using our Applied Biosystems™ GeneChip™ Arrays. The signatures were further verified by testing samples from lipopolysaccharide (LPS)-treated mice with QuantiGene Plex and qPCR assays. The spleens from 52 treated and untreated mice were harvested to prepare tissue lysates for the QuantiGene Plex Assay or to purify RNA for qPCR testing. Both the QuantiGene Plex and qPCR assays showed similar patterns of up- and down-regulation upon treatment. Data courtesy of Amgen, Inc [2].
Benefit: The ability to test simple lysates makes the QuantiGene Plex Assays robust, accurate, and precise. Multiplexing increases efficiency and conserves sample.
7
Application 4: Verification of GeneChip array signatures from FFPEUsing Applied Biosystems™ GeneChip™ Human Exon 1.0 ST Arrays, 19 cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples were screened to find a gene signature that would differentiate SCC from AC. The FFPE blocks had been stored for an average of 12 (range 10–16) years. After analysis of the exon array data, a QuantiGene 26-plex panel was constructed and successfully verified against the 19 cervical SCC and 9 cervical AC FFPE samples. Reprinted with permission from Macmillan Publishers Ltd on behalf of Cancer Research UK [3].
Benefit: Formalin has been known to degrade and chemically modify RNA, which can be an issue for assays that use DNA polymerases as part of their template amplification step. QuantiGene Plex Assays work well with FFPE samples because the assay is based on hybridization. The assay works directly with FFPE tissues without the need for RNA purification or amplification.
SKBR3HeLa
HER2 = 2 HER2 = 14
0
1
2
3
4
5
6
7
8
9
MAPKAPKCh 1
MYSTCh 5
BRFCh 8
FGFRCh 8
ERBBCh 17
GRBCh 17
PNMTCh 17
SMARCE1Ch 17
No
rmal
ized
to
ski
n �b
rob
last Normal skin fibroblast
MDA-231
SKBR3ERBB2 (HER2)/normal
Ratio = 6.5
SKBR3HeLa
HER2 = 2 HER2 = 14
0
1
2
3
4
5
6
7
8
9
MAPKAPKCh 1
MYSTCh 5
BRFCh 8
FGFRCh 8
ERBBCh 17
GRBCh 17
PNMTCh 17
SMARCE1Ch 17
No
rmal
ized
to
ski
n �b
rob
last Normal skin fibroblast
MDA-231
SKBR3ERBB2 (HER2)/normal
Ratio = 6.5
Application 5: DNA breakpoint analysisThe Invitrogen™ QuantiGene™ Plex DNA Copy Number Assay was used to detect DNA breakpoints for HER2 and adjacent genes on chromosome 17, as well as control genes on chromosomes 1, 5, and 8 in SKBR3 breast cancer cells and control cell lines (normal skin fibroblasts and MDA-231 cells). As expected, a 7-fold amplification (6.5 normalized ratio) of HER2 was detected. The QuantiGene Plex Assay result was concordant with that of a bDNA fluorescence in situ hybridization (FISH) assay (Note: the DNA assay is not currently commercially available), in which 14 copies can be counted in SKBR3 cells vs. 2 copies in the HeLa cells as expected, again showing a 7-fold amplification. Amplification of PNMT6 and GRB7, 2 genes adjacent to HER2, were also detected at 7- to 8-fold, whereas control genes were detected at the expected single-copy number.
Benefit: The QuantiGene Plex DNA Assay has the ability to interrogate single-copy DNA changes across a given chromosome to further analyze DNA breakpoint changes. The assay has the ability to measure up to 33 regions per well of a 96-well plate. Color key
Row z-score
SCC
AC
p63+
p63–
−3 −2 −1 0 1 2 3
S100A8KRT5CTA-55I10.1TRIM29S100A2FAT2CLCA2DSG3DSC3TP63CSTADENND2CUSH1CTSPAN8KIF12HGDAC105391.3PROM1DDAH1SPINK1EPS8L3RGL3IL20RBSLC44A4TSPAN12TFF3
V554
V553
V558
V611
B
V562
V568
V572
V636
V629
V509
V622
V615
V524
V669
-2
V548
V545
V505
Histology
p63
8
6mm
6 mm6 mm
Liver FFPE sectionsSection 1
Section 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Mea
n �u
ore
scen
ce in
tens
ity
Genes
FFPE liver sections—1 vs. 2
0
400
800
1,200
1,600
6 mm
H&E-stainedsection 1
Unstainedsection 2
6mm
6 mm6 mm
Liver FFPE sectionsSection 1
Section 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Mea
n �u
ore
scen
ce in
tens
ity
Genes
FFPE liver sections—1 vs. 2
0
400
800
1,200
1,600
6 mm
H&E-stainedsection 1
Unstainedsection 2
Application 7: H&E-stained and unstained FFPE slidesGene expression analysis was performed on H&E-stained and unstained FFPE tissue sections. Two slides were obtained and a 6 x 6 mm portion of tissue (5 µm thick) was removed from each slide and homogenized in the QuantiGene lysis buffer. A 23-plex gene panel consisting of 20 target genes and 3 housekeeping genes was used with the 2 samples. Comparable expression was observed in both sections for all 23 genes with an overall CV of 8.6%.
Benefit: The assay allows the use of H&E-stained slides. In this example, a 6 x 6 mm size section of tissue provides users with the ability to test small samples in previously stained H&E-stained slides. Areas of interest identified in H&E-stained FFPE sections are viable samples for subsequent expression analysis.
Clofibrate
0
10
20
30
Fo
ld in
du
ctio
n vs
. veh
icle
co
ntro
l
Fold induction of mRNA
Rifampin
3-MethylcholanthrenePhenobarbitalRitonavirOmeprazolePhenytoinß-Napthoflavone
mRNA CYP panel
1A1 1A2 1E1 2A6 2C6 2C9 2C18 2C19 2B6 2D6 2E1 3A4 3A5
Application 6: ADME and toxicology screeningTranscripts for 13 different cytochrome p450 genes were measured in toxicant-treated human hepatocytes at a single dose. Cryopreserved human donor hepatocytes were cultured for 2 days followed by treatment with 1 of the 8 toxicants or vehicle control. After 48 hours, the cells were lysed using the Invitrogen™ QuantiGene™ Plex lysis reagent, and the lysate was used directly in the QuantiGene Plex Assay. The investigators also found that there was a strong correlation between the levels of mRNA expression and the levels of enzyme activity of the encoded proteins. Data courtesy of Genentech [4].
Cryopreserved hepatocytes from a single donor were treated with omeprazole and dosed between 150 and 0.21 μM. A QuantiGene 18-plex panel consisting of CYP and transporter genes were used to investigate the mRNA response. The EC50 calculations and the dose response curves were prepared for the selected CYP and transporter genes. Data courtesy of Celsis IVT [5].
Benefit: The protocol for the QuantiGene Plex Assay is invariant regardless of the panel of genes that is measured. Unlike enzyme assays, no further optimization is required when going from 1 panel to the next. The specificity of QuantiGene Plex Assays allows closely related genes to be measured simultaneously in the same well.
Fold
ind
uctio
n
SLC01B3
log (M)
0.0
0.2
0.4
0.6
0.8
1.0
-8 -7 -6 -5 -4 -3
Fold
ind
uctio
n
SLC22A1
log (M)-8 -7 -6 -5 -4 -3
0.0
0.5
1.0
1.5
Fold
ind
uctio
n
UGT2B7
log (M)
0.0
0.2
0.4
0.6
0.8
1.0
-8 -7 -6 -5 -4 -3
Fold
ind
uctio
n
UGT1A4
log (M)-8 -7 -6 -5 -4
0
1
2
3EC50 1.063e–005
Fold
ind
uctio
n
CYP3A4
log (M)
0.0
0.5
1.0
1.5
2.0
2.5
-8 -7 -6 -5 -4
EC50 8.585e–006
Fold
ind
uctio
n
CYP2B6
log (M)
EC50 1.676e–005
-8 -7 -6 -5 -40
1
2
3
4
Fold
ind
uctio
n
CYP1A2
log (M)
0
2
4
6
8
10
-8 -7 -6 -5 -4
EC50 2.008e–006
Omeprazole dose response curve
9
Application 8: Classification of melanoma subgroups using QuantiGene Plex gene signatures62 genes relevant to melanoma were incorporated in a QuantiGene Plex Assay as a proof of concept to demonstrate the capability to discover biomarkers. Tissue lysates were prepared from frozen sections from 20 cases of metastatic melanoma alongside the corresponding normal skin counterparts. Data analysis identified those genes most closely linked with the disease.
Gene expression measurements were made against the 62 genes, and each gene of interest was normalized by the geometric mean of 5 housekeeping genes. The P value for each gene was calculated using a supervised Student’s t-test. The genes with the best 7 P values were analyzed using an unsupervised principle component analysis (PCA).
In situ gene analysis of these genes was performed using QuantiGene ViewRNA analysis. Metastatic and normal skin sections were probed for the transcripts of KRT5, CXCL12, ARPC2, and PCNA genes. The dye used in the assay is colorimetric but can also be viewed under fluorescence. Data courtesy of California Pacific Medical Center/UCSF [6].
Benefit: Ability to work directly with melanoma tissues without RNA purification or concerns about melanin inhibition of DNA polymerase.
–5 1050
–4
–2
0
2
4
pca2$x[.1]
pca
2$x[
.2]
Gene P value
BCL6 1.28 x 10–9
PTEN 1.03 x 10–8
ARPC2 2.17 x 10–8
CXCL12 1.37 x 10–7
BRAF 4.18 x 10–7
PCNA 6.33 x 10–7
CLEC3B 1.13 x 10–6
Metastatic melanomaNormal PCA
Principal component analysis of 7 genes
M17
M17: Not melanoma; lymph node with pigmented histiocytes
10
6
0
1
2
3
4
5
7
CD86 IL2RACCL8IL8CXCL5 Cig5CXCL10TNFalP6IFNGIL1R1 IFIT3
Rat
io o
f CXCL10
-tre
ated
to
unt
reat
ed
CXCL10 stimulation of whole blood for 6 hr and QuantiGene analysis of gene expression
0.3 µg CXCL10
1.0 µg CXCL10
1.0 µg CXCL10 Heat-inactivated
Application 9: Stimulation of whole human bloodCXCL10-responsive gene induction was confirmed using a QuantiGene 12-plex panel to test RNA isolated from healthy donor, blood-treated ex vivo with CXCL10. Gene induction was shown to be dose-dependent and was eliminated upon heat inactivation of the chemokine. Gene expression was normalized to the expression level of the GAPDH housekeeping gene and presented as the ratio of treated:untreated. Data courtesy of Medarex Inc, a BMS Company [7].
Benefit: QuantiGene Plex Assays work directly with whole blood without the need for globin depletion or RNA purification.
Limit of detection ≤1,000–2,000 transcripts/assay well
Limit of quantitation ≤2,000–4,000 transcripts/assay well
Linear dynamic range ≥3.0 logarithmic units
Assay CV ≤15% intra-assay; ≤20% inter-assay
Compatible sample typesCultured cells, bacteria, whole bood, PAXgene, Invitrogen™ Tempus™ samples, dried blood spots, fresh-frozen tissues (animal or plant), FFPE samples, purified RNA
Assay format 96- or 384-well plate
Targets/well 3–80 for RNA; 3–33 for DNA
SpecificationsThe QuantiGene Plex Assay can be used for both mRNA profiling and DNA copy number variation.
11
Off-the-shelf panelsWe have also developed over 3,700 panels that cover over 17,000 genes. These available panels cover a wide range of host species that include: human, mouse, rat, canine, monkey, porcine, soy, maize, and many others. Any combination of existing genes can be combined to create a new panel. To find the latest panels and genes, please go to thermofisher.com/quantigene or contact one of our probe designers at [email protected]
References1. Beer DG et al. (2002) Gene expression profiles predict survival of patients with lung
adenocarcinoma. Nat Med. 8:816-824.
2. Ebsworth K (2007) Gene expression analysis by Quantigene Plex: validation and applications. Paper presented at: Planet xMAP USA; 2007 March 12-14; Laguna Hills, CA.
3. Hall JS et al. (2011) Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples. Br J Cancer 6:971-981.
4. Wong S et al. New methodology for measuring mRNA levels along with monitoring CYP activity in induction studies using human plateable cryopreserved hepatocytes. Paper presented at: The 8th International Society for the Study of Xenobiotics; 2007 Oct 9-12; Sendai, Japan.
Human Apoptosis 80-plex panelCASP6 CARD8 BNIP2 DAPK1 BAG3 HPRT1 TNFRSF10A BID TNFRSF7 LTBR TNFRSF9 BIRC8 CASP3 TP53 BIRC3 TNFRSF1A
PYCARD AKT1 BRAF TP53BP2 BAD BCL2L1 TP73 GADD45A FADD TNFRSF21 BCL2L2 NOL3 FASLG CASP8 PPIB CASP4
CASP1 CASP2 BCLAF1 ABL1 TNF TNFSF10 TNFRSF11B APAF1 TNFSF8 CASP14 IGF1R BCL10 LTA TNFRSF10B CASP9 BCL2L11
TRAF4 BFAR MCL1 BIRC6 TNFSF5 BAG1 BCL2A1 BAK1 CIDEB TRAF2 TRAF3 CFLAR CASP10 CD40 BNIP3L RIPK2
CASP7 BAG4 BIRC1 BCL2L10 TRADD BNIP1 TNFRSF25 BAX BNIP3 BIRC2 CARD4 CARD6 CIDEA FAS BCL2 BIK
Human Cancer 80-plex panelNUSAP1 ERBB3 WNT2 SPARC RAB17 PRAME GJB1 SLC7A1 MCAM RNF157 ACSL3 TRPM4 CACNA1D TOP2A BRAF SPARCL1
MYLK BIRC5 ARPC2 CDKN1B POU2F3 CCNE1 FN1 E2F1 ZNF577 PHIP HIF1A KIF20A MAGEA1 C10orf137 HPRT1 BUB1
CASP8 KLK3 CDKN2B CCND1 RB1 CDH2 YWHAZ MCM6 DSC1 MCM4 MITF VCAN SPP1 MKI67 MYRIP PCSK6
TP53 NCOA3 PCNA TFRC F10 ITGB1 CCR7 PGK1 PTEN ITGB3 MMP10 MMP9 GSTP1 MMP2 MLANA BCL2A1
RAD54B MET GCNT1 AQP3 MCM10 WNT5A DCT TBP GPC3 CDKN2A PRKCA ITGB4 CRISP3 CCNA1 GUSB PLAUR
Human Cytokine 80-plex panelIL1F6 IL1F7 IFNA5 IFNA8 IL1F8 IFNB1 IL19 CD70 IL3 IL1F10 IL17E FAM3B NODAL TNFSF13 GDF3 TNFSF4
BMP4 INHBA TNFSF12 BMP2 TGFB3 CSF1 BMP1 BMP6 GDF2 GDF5 GDF9 BMP7 TXLNA BMP3 TNFSF13B IL16
TNFSF14 IL1F5 GDF10 BMP8B IL24 IL9 TNF IL12A IL22 IL13 IL17A TNFSF10 TNFSF11 TGFB1 IL7 TGFA
PDGFA IL12B TGFB2 TNFRSF11B IL10 BMP5 IL5 GAPDH IL18 GDF11 IFNA2 IL2 CSF2 IL20 FASLG IL1F9
IFNA1 IL4 IFNG LTA IL6 FIGF ACTB IL1B IL1A GDF8 IL15 LTB IFNK PPIB IL8 IL21
Human Inflammation 80-plex panelPYCARD MAPK3 SUGT1 MAP3K7IP1 MAP3K7 MEFV MAPK12 PSTPIP1 MAPK13 TNFSF14 PEA15 RIPK2 PANX1 NLRX1 NALP1 NLRP9
TNFSF4 P2RX7 NLRP4 MAP3K7IP2 BIRC1 NLRC5 BIRC4 NLRP3 NLRP5 AIM2 BIRC2 CARD6 CASP1 CASP5 CHUK CIITA
CTSB HSP90AA1 TRA1 IKBKB IKBKG IRF2 MAPK9 NFKB1 MAPK8 NFKBIA RAGE TNF TNFSF5 IL12A BCL2 TNFSF11
ICEBERG CASP4 IRF1 IL12B MAPK1 GAPDH CXCL1 CASP8 TRAF6 CXCL2 CCL5 CCL7 RELA PTGS2 IFNG IL6
TIRAP IRAK1 ACTB IL1B TXNIP MYD88 CARD12 BIRC3 CCL2 PPIB IFNB1 NOD2 IL18 BCL2L1 HSPCB NALP12
Below are some examples of the genes included in the 80-plex panelsThe list of our 80-plex panels as well as the rest of our 3,700 panels can be found on our website at thermofisher.com/quantigene
5. Moeller T et al. Celsis—IVT − Transcript regulation of 18 ADME genes by prototypical inducers in human hepatocytes. Paper presented at: The 18th International Society for the Study of Xenobiotics. 18th North American Regional Meeting; 2012 Oct 14-18; Dallas, TX.
6. Leong SP et al. Paper presented at: The 4th International Symposium on Cancer Metastasis and the Lymphovascular System: Basis for Rational Therapy; 2011 May 12-14; New York, NY.
7. Witte A et al. Medarex—CXCL10 expression and biological activities in inflammatory bowel disease. Paper presented at: The Digestive Disease Week Meeting; 2008 May 19-21; San Diego, CA.
Custom panelsCustom Quantigene Plex panels are available for any gene and any species. Custom panels ranging from 3- to 80-plex can be designed and shipped within 2 weeks. Customers simply provide us with a gene list, RefSeq IDs, or DNA sequences.
For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. VERSANT is a trademark of Siemens. PAXgene is a trademark of PreAnalytiX GmbH. Luminex, xMAP, MAGPIX, and FLEXMAP 3D are registered trademarks, and 100/200 are trademarks of Luminex Corporation. JAX is a trademark of The Jackson Laboratory. COL31609 0417
Find out more and place your order at thermofisher.com/quantigene
QuantiGene Plex panels—pathway guide
QuantiGene Plex pathway panels
Hum
an
Mo
use
Rat
Apoptosis and autophagy
Apoptosis • • •
Autophagy • •
Unfolded protein response • •
Cancer signaling
Angiogenesis • • •
Angiogenic growth factors and angiogenesis inhibitors • • •
Breast cancer and estrogen receptor signaling • • •
Cancer pathway • • •
Cancer drug resistance and metabolism • •
Cell cycle • • •
Epithelial to mesenchymal transition (EMT) • •
Extracellular matrix and adhesion molecules • • •
Oncogenes and tumor suppressor genes • •
Tumor metastasis • • •
Cardiology and bone biology
Atherosclerosis • •
Osteogenesis • • •
Skeletal muscle—myogenesis and myopathy • • •
Epigenetics
Epigenetic chromatin modification enzymes • •
Epigenetic chromatin remodeling factors • •
Polycomb group (PcG) genes and DNA methylation • •
Transcription factors • •
Inflammation and immunology
Cell surface markers •
Chemokines and receptors • • •
Cytokines • • •
Cytoskeleton regulators • •
Dendritic and antigen-presenting cell • •
HIV infection and host response •
Inflammation • •
Inflammasomes • • •
Inflammatory cytokines and receptors • • •
Inflammatory response and autoimmunity • • •
Innate and adaptive immune responses • •
Interferon α and β response • •
Interferon and receptor •
Interferon (IFN) and receptor mouse •
T cell and B cell activation • • •
T cell activation • •
T cell anergy and immune tolerance • •
Th1-Th2-Th17 • • •
Th17 for autoimmunity and inflammation • • •
T helper cell differentiation • •
Toll-like receptor signaling pathway • • •
Tumor necrosis factor (TNF) ligand and receptor • •
QuantiGene Plex pathway panels
Hum
an
Mo
use
Rat
Metabolism and endocrinology
Adipogenesis • • •
Fatty acid metabolism • •
Glucose metabolism • • •
Diabetes • • •
Glycosylation • •
Insulin signaling pathway • • •
Mitochondria • • •
Mitochondrial energy metabolism • •
Hypertension • • •
Obesity • • •
Neuroscience
Neurogenesis and neural stem cell • • •
Neuroscience ion channels and transporters • • •
Neurotransmitter receptors and regulators • • •
Neurotrophin and receptors • • •
Stem cells
Embryonic stem cells • •
Endothelial cell biology • • •
Hematopoietic stem cells and hematopoiesis • •
Mesenchymal stem cell • •
Stem cell • • •
Stem cell signaling • •
Terminal differentiation markers • •
Toxicity and drug metabolism
Cardiotoxicity • • •
Drug metabolism • • •
Drug metabolism phase I enzymes • •
Drug metabolism phase II enzymes • •
Drug transporters • • •
Hepatotoxicity • • •
Lipoprotein signaling and cholesterol metabolism • • •
Nephrotoxicity • • •
Neurotoxicity • • •
Stress and toxicity • • •
Toxicity •
Housekeeping genes
Housekeeping genes • • •
Custom
Any gene for any model organism • • •
