QuantiGene assays for analysis of expression or copy number variation

Quantitate up to 80 from cell lysates or tissue homogenates QuantiGene Singleplex Assays

Accurate and precise RNA quantitation in a simple workflow

QuantiGene Singleplex Assays Key benefits By quantitating mRNA or DNA directly • Fast sample prep—perform the assay directly on cell lysates or tissue from your starting sample and using a homogenates, no need for RNA purification signal amplification assay, Invitrogen™ ™ • —detect changes smaller QuantiGene Singleplex Assays are Precisely detect subtle changes than 10% an accurate and precise method of quantitating gene expression with a • Works with difficult sample types—easily quantitate heavily degraded and simple workflow. cross-linked RNA in formalin-fixed, paraffin-embedded (FFPE) tissues, or quantitate RNA in blood Technical overview and workflow QuantiGene assays utilize branched • Quick turnaround—thousands of probe designs stocked; custom probes DNA (bDNA) technology that has been designed to any sequence, and ready to ship within a few days used for decades in the VERSANT™ • Superior specificity—delivers greater specificity than other common 3.0 diagnostic assays for HIV-1, HCV, technologies, and distinguishes between closely related genes due to probe and HBV. First, cells are lysed or design with several capture points along the target mRNA of interest tissue samples are homogenized to release the target RNA. Second, the • Flexibility—easily automated for use in routine compound screening oligonucleotide probe set is incubated with the target RNA and capture plate • Compatibility—works with a variety of sample types: cultured cells, whole overnight. During this incubation, the blood, PAXgene™ blood, dried blood spots, fresh or frozen animal or plant probes cooperatively hybridize to the tissues, FFPE samples, and purified RNA, using standard instrumentation target and capture probes bound to (microplate luminometer and a horizontal airflow oven) the plate, capturing the target RNA. Sample Target Signal Third, signal amplification is performed Detection preparation hybridization amplification via sequential hybridization of the bDNA preamplifier, amplifier, and Chemiluminescent Capture Preamplifier label-probe molecules to the target. extender (CE) substrate

Addition of a chemiluminescent Na O PO S Cl Label Amplifier 2 3 extenders (LE) substrate generates a luminescence N CH signal directly proportional to the Blocking Label probe 3 probe (BL) amount of target mRNA present in Lysate the sample. (with target Hybridizations Read signal with RNA) and wash steps a luminometer

Incubate capture plate with sample and probes

Lyse sample

Na2O3PO S Cl Na2O3PO S Cl

N N

CH3 CH3

Lysate (with target RNA) Capture plate Capture probe

2 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.

140 Exceptional accuracy of measurement— 120 spike recoveries of 85–115%

100 A wide range of concentrations (0.001–1.0 attomoles) of an I L- 6 in vitro–transcribed (IVT) RNA was spiked into a cell 80

Recovery (%) lysate with undetectable levels of endogenous expression. 60 0.000 0.001 0.010 0.100 1.000 Percent recovery of the spike was calculated based on the IL6 IVT RNA (attomole) signal from the IVT RNA in the presence of lysate.

350-fold induction 7.000 siRNA knockdown screening and verification

6.000 Phorbol myristate acetate (PMA) induction of HeLa cells led

5.000 to a spike in IL-8, which was then knocked down by siRNA Δ7% 4.000 treatment. Knockdown efficiency was measured at two

3.000 87% knockdown 94% knockdown time points and a 7% change was accurately detected by

2.000 QuantiGene assays.

1.000 Ratio (IL-8:cyclophilin) Ratio 0.000 Untreaded PMA-induced IL-8 siRNA IL-8 siRNA HeLa HeLa 4 hr post- 8 hr post- treatment treatment

80 Detection of low-abundance genes Background 70 QuantiGene assays were able to detect RNAs from 3 Brain RNA 60 low-abundance genes in two reference samples: brain Universal Human Reference RNA 50 RNA and universal human reference RNA. These genes 40 were undetectable using other technologies. This exquisite 30 sensitivity allows for the basal measurement of low- 20 expression genes. 10

Relative luminescence unit (RLU) unit luminescence Relative 0 C11 or f9 GULP1 RECQL4 10 ng/well

3N 3T 14N 14T Quantitative gene expression data from archived 5 FFPE samples 3T/3N 28S 4 Profiling with a QuantiGene Singleplex Assay for lactate 14T/14N 3 dehydrogenase RNA in 14-year old FFPE lung tumor (14T) 18S 2 and adjacent normal tissue (14N) demonstrated a 2–3 fold Fold change Fold induction of this advanced-stage cancer biomarker, in 1 agreement with published data [1]. 0 LDHA RPL19 RPL32 RNA from 3- and 14-year old FFPE In agreement with the literature samples of tumor (T) and adjacent [1], QuantiGene assay detected normal (N) tissue from lung cancer a 2-fold induction of LDHA RNA patients as visualized by agarose gel in tumor relative to the normal electrophoresis. The positions of the tissue, even in highly degraded 28S and 18S RNAs are indicated. 14-year old samples.

3 QuantiGene Plex Assays

Accurate and precise RNA quantitation of up to 80 genes in a single well

Overview of QuantiGene Key features and benefits of the assay Plex Assays • True multiplexing—measure up to 80 genes of interest and housekeeping The Invitrogen™ QuantiGene™ Plex genes in the same well with no cross-reactivity Assays provide an accurate and • —96-well plate format compatible with Luminex® precise method for multiplexed Standardized platform ™ ® ® gene expression quantitation. 100/200 , MAGPIX , and FLEXMAP 3D systems ® ® Using Luminex xMAP technology, • Simple workflow—ELISA-like workflow for direct hybridization of transcripts QuantiGene Plex Assays allow for to beads and transcript labeling simultaneous measurement of 3–80 mRNA species in every well of a • Works with difficult sample types—works with degraded and crosslinked 96-well plate. QuantiGene Plex Assays RNA in FFPE tissues, and directly with blood also incorporate the exclusively • Large inventory of verified genes—over 15,000 genes can be mixed to licensed branched DNA technology create pathway- and disease-themed panels from Siemens. Branched DNA assays allow for the direct measurement • Fast customization—if we don’t have your gene(s), we can create and QC of RNA transcripts by using signal your custom panel within 2 weeks amplification rather than template amplification. • ISH compatible—same technology utilized in the Invitrogen™ QuantiGene™ ViewRNA™ Kits for mRNA in situ hybridization (ISH) 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 Sample Target Signal ™ Detection for verifying Applied Biosystems preparation hybridization amplification GeneChip™ analysis or next-generation Luminex beads Streptavidin with capture probes sequencing (NGS) data and for Preamplifier phycoerythrin (SAPE) verifying biomarkers for translational research. Amplifier Read signal using a Luminex instrument Label probe Incubate beads with sample and probes 80 RNA targets per well Hybridizations Capture and wash steps extenders (CE) Lyse sample

Label extenders (LE)

Blocking Lysate probes (BL) (with target RNA)

Lysate (with target RNA)

4 QuantiGene Plex Assay applications QuantiGene 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.

101

10 0

Target discoveryqPCR 10−1 Lead optimization Preclinical studies Clinical studies ∆∆CT –

assay 2 Target identification and 10−2 High-throughput screening ADME Biomarker verification verification of cell-based microarrays10−3 Secondary screening Toxicology Clinical trials (FFPE tissues) 10−4 p40 p35 TNF p19 EB13 p28 LPS + LPS Liver FFPE sections CXCL10 stimulation of whole blood for 6 hrFFPE and QuantiGene liver analysis sections—1 of gene expression vs. 2 0 10 H&E-stained Unstained 7 Section 1 0.3 µg CXCL10 – section 1 section 2 1,600 6 1.0 µg CXCL10 9 10 ¹ Normal skin fibroblast HeLa SKBR3 1.0 µg CXCL10 Section 2 Heat-inactivated 8 MDA-231 ERBB2 (HER2)/normal 5 7 – SKBR3 Ratio = 6.5 QuantiGene 10 ² 6 1,200 4 5

Plex Assay – 4 untreated to treated 10 ³ 3 3

2 6mm 6 mm 800 10 –4 1 2

Normalized to skin broblast 0 atio o CXCL10 Fluorescence/µg RNA Fluorescence/µg MAPKAPK MYST BRF FGFR ERBB GRB PNMT SMARCE1 1 Ch 1 Ch 5 Ch 8 Ch 8 Ch 17 Ch 17 Ch 17 Ch 17 6 mm –5 10 400 6 mm 0 p40 p35 TNF p19 EB13 p28 HER2 = 2 HER2 = 14 CD86 CXCL5 IL1R1 IL8 IFNG CCL8 TNFP6 IL2RA CXCL10 IFIT3 Ci5

Mean uorescence intensity uorescence Mean 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Patient-derived xenograft Verification of genes DNA copy number Verification of genes Expression analysisGenes (PDX) models involved with inflammation for breakpoint analysis in FFPE samples from whole human blood

Color key

Basal Luminal −3 −2 −1 0 1 2 3 Row z-score Principal component analysis of 7 genes SCC AC p63+ 4 p63– Fold induction of mRNA Metastatic melanoma Gene P value Histology p63 Normal PCA S100A8 BCL6 Rifampin

HCC70 HCC3153 MDAMB468 SUM1314 BT20 H5578T BT549 MDAMB157 MDAMB231 MDAM453 LY2 SUM185PE T47D HCC202 BT474 ZR751 MDAMB361 AU565 SKBR3 KRT5 30 Clofibrate CTA55I101 2 TRIM29 3-Methylcholanthrene PTEN S100A2 Phenobarbital FAT2 Ritonavir CLCA2 Omeprazole ARPC2 DSG3 20 DSC3 Phenytoin 0 TP63 ß-Napthoflavone EN1 CSTA CXCL12 DENND2C FOXC1 USH1C pca TSPAN8 10 –2 BRAF GABRP KIF12 HGD AC1053913 ERBB2 PROM1 PCNA DDAH1 CA12 SPINK1 0 –4 EPS8L3 control veicle vs induction old 1A1 1A2 1E1 2A6 2C6 2C9 2C18 2C19 2B6 2D6 2E1 3A4 3A5 CLEC3B AGR2 RGL3 IL20RB TFF3 SLC44A4 m CYP panel TSPAN12 –5 0 5 10 GATA3 TFF3 ot melanoma lymp node it pigmented istiocytes V615 V524 V572 V622 V629 V562 V558 V553 V568 V554 V505 V636 V548 V545 ESR1 V509 pca V611B V6692 SCNN1 Tumor cell line characterization FFPE analysis of genes CYP genes used with PCA analysis of genes for drug compound screening involved with cancer human hepatocytes involved with melanoma

Application 1: Patient-derived xenograft (PDX) model mean fluorescence intensity values for the 6 genes of A 10-plex QuantiGene Plex panel was developed to interest that were obtained with a Luminex® instrument. simultaneously measure the expression of human and The data demonstrated assay specificity: human samples mouse genes in a PDX model. The panel comprised 3 were negative for mouse transcripts while untreated mice human genes of interest and 2 human housekeeping samples were negative for human transcripts. As expected, genes, as well as 3 mouse genes of interest and 2 tissues from xenografted mice contained both human and mouse housekeeping genes. Patient-derived tumors murine transcripts. were implanted into JAX™ NOD scid gamma (NSG) mice. After growth and metastasis of the tumor in mice, both Benefit: The specificity of each component assay the liver and spleen from the mice were harvested, and within a customized multiplexed QuantiGene Plex panel lysates from untreated and xenografted mice were tested allows the assay to be tailored to address complex alongside human liver RNA. The table below shows the biological questions.

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 7,6 55 1 18,023 4 Mouse liver 392 4 1,488 2 26,586 2 Xenograft spleen 3,321 27,6 42 472 24,605 24,563 6,547 Xenograft liver 2,959 21,790 2,114 7,0 58 27,826 1,702

5 Application 2: Cell line characterization for Application 3: Target verification compound screening Gene expression signatures for inflammation pathway Breast cancer cell lines were created from primary tumors that activation were identified using our Applied Biosystems™ contained recurrent genetic abnormalities. A QuantiGene GeneChip™ Arrays. The signatures were further verified by 12-plex assay was developed to differentiate patterns of RNA testing samples from lipopolysaccharide (LPS)-treated mice expression across the cell lines. When the normalized levels with QuantiGene Plex and qPCR assays. The spleens from of gene expression were plotted on a heat map, the data 52 treated and untreated mice were harvested to prepare showed that the pattern of expression for the 12 genes could tissue lysates for the QuantiGene Plex Assay or to purify be used to classify the cell lines as being either the basal or RNA for qPCR testing. Both the QuantiGene Plex and the luminal subtype of breast cancer. The cell lines were then qPCR assays showed similar patterns of up- and down- used for further screening of drug compounds based on the regulation upon treatment. Data courtesy of Amgen, Inc [2]. specific subtypes. Data courtesy of Joe Gray and Nick Wang, Oregon Health Sciences University. Benefit: The ability to test simple lysates makes the QuantiGene Plex Assays robust, accurate, and precise. Benefit: Multiplexing allows for rapid classification of cell Multiplexing increases efficiency and conserves sample. lines that can then be used for compound screening.

101

Basal Luminal 10 0

qPCR 10−1 ∆∆CT – assay 2 10−2

10−3 HCC70 HCC3153 MDAMB468 SUM1314 BT20 H5578T BT549 MDAMB157 MDAMB231 MDAM453 LY2 SUM185PE T47D HCC202 BT474 ZR751 MDAMB361 AU565 SKBR3

10−4 p40 p35 TNF p19 EB13 p28 LPS + LPS EN1 10 0 FOXC1 GABRP 10 –¹ ERBB2 – CA12 QuantiGene 10 ² Plex Assay AGR2 10 –³ TFF3 GATA3 10 –4 ESR1 RNA Fluorescence/µg 10 –5 SCNN1 p40 p35 TNF p19 EB13 p28

6 Application 4: Verification of GeneChip array Application 5: DNA breakpoint analysis signatures from FFPE The Invitrogen™ QuantiGene™ Plex DNA Copy Number Using Applied Biosystems™ GeneChip™ Human Exon 1.0 Assay was used to detect DNA breakpoints for HER2 and ST Arrays, 19 cervical squamous cell carcinoma (SCC) and adjacent genes on 17, as well as control 9 adenocarcinoma (AC) FFPE samples were screened to genes on 1, 5, and 8 in SKBR3 breast find a gene signature that would differentiate SCC from cancer cells and control cell lines (normal skin fibroblasts AC. The FFPE blocks had been stored for an average of and MDA-231 cells). As expected, a 7-fold amplification (6.5 12 (range 10–16) years. After analysis of the exon array normalized ratio) of HER2 was detected. The QuantiGene data, a QuantiGene 26-plex panel was constructed and Plex Assay result was concordant with that of a bDNA successfully verified against the 19 cervical SCC and 9 fluorescence in situ hybridization (FISH) assay (Note: cervical AC FFPE samples. Reprinted with permission from the DNA assay is not currently commercially available), Macmillan Publishers Ltd on behalf of Cancer Research in which 14 copies can be counted in SKBR3 cells vs. UK [3]. 2 copies in the HeLa cells as expected, again showing a 7-fold amplification. Amplification of PNMT6 and GRB7, 2 Benefit: Formalin has been known to degrade and genes adjacent to HER2, were also detected at 7- to 8-fold, chemically modify RNA, which can be an issue for assays whereas control genes were detected at the expected that use DNA polymerases as part of their template single-copy number. amplification step. QuantiGene Plex Assays work well with FFPE samples because the assay is based on Benefit: The QuantiGene Plex DNA Assay has the ability hybridization. The assay works directly with FFPE tissues to interrogate single-copy DNA changes across a given without the need for RNA purification or amplification. chromosome to further analyze DNA breakpoint changes. The assay has the ability to measure up to 33 regions per Color key well of a 96-well plate. −3 −2 −1 0 1 2 3 Row z-score

9 SCC Normal skin fibroblast HeLa SKBR3 AC 8 MDA-231 + ERBB2 (HER2)/normal p63 7 p63– SKBR3 Ratio = 6.5 Histology 6 p63 5 S100A8 KRT5 4 CTA55I101 TRIM29 3 S100A2 2 FAT2 CLCA2 1 DSG3

DSC3 Normalized to skin broblast 0 TP63 MAPKAPK MYST BRF FGFR ERBB GRB PNMT SMARCE1 CSTA Ch 1 Ch 5 Ch 8 Ch 8 Ch 17 Ch 17 Ch 17 Ch 17 DENND2C USH1C TSPAN8 HER2 = 2 HER2 = 14 KIF12 9 Normal skin fibroblast HGD HeLa SKBR3 8 MDA-231 AC1053913 ERBB2 (HER2)/normal PROM1 7 SKBR3 Ratio = 6.5 DDAH1 6 SPINK1 EPS8L3 5 RGL3 4 IL20RB SLC44A4 3 TSPAN12 2 TFF3 1 V615 V524 V572 V622 V629 V562 V558 V553 V568 V554 V505 V636 V548 V545 V509 V611B V6692 Normalized to skin broblast 0 MAPKAPK MYST BRF FGFR ERBB GRB PNMT SMARCE1 Ch 1 Ch 5 Ch 8 Ch 8 Ch 17 Ch 17 Ch 17 Ch 17

HER2 = 2 HER2 = 14

7 Application 6: ADME and toxicology screening Application 7: H&E-stained and unstained FFPE slides Transcripts for 13 different cytochrome p450 genes were Gene expression analysis was performed on H&E-stained measured in toxicant-treated human hepatocytes at a and unstained FFPE tissue sections. Two slides were single dose. Cryopreserved human donor hepatocytes obtained and a 6 x 6 mm portion of tissue (5 µm thick) were cultured for 2 days followed by treatment with 1 of the was removed from each slide and homogenized in the 8 toxicants or vehicle control. After 48 hours, the cells were QuantiGene lysis buffer. A 23-plex gene panel consisting of lysed using the Invitrogen™ QuantiGene™ Plex lysis reagent, 20 target genes and 3 housekeeping genes was used with and the lysate was used directly in the QuantiGene Plex the 2 samples. Comparable expression was observed in Assay. The investigators also found that there was a strong both sections for all 23 genes with an overall CV of 8.6%. correlation between the levels of mRNA expression and the levels of enzyme activity of the encoded . Data Benefit: The assay allows the use of H&E-stained slides. courtesy of Genentech [4]. In this example, a 6 x 6 mm size section of tissue provides users with the ability to test small samples in previously Cryopreserved hepatocytes from a single donor were stained H&E-stained slides. Areas of interest identified treated with omeprazole and dosed between 150 and in H&E-stained FFPE sections are viable samples for 0.21 μM. A QuantiGene 18-plex panel consisting of CYP subsequent expression analysis. 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 Liver FFPE sections FFPE liver sections—1 vs. 2 genes. Data courtesy of Celsis IVT [5]. H&E-stained Unstained Section 1 section 1 section 2 1,600 Section 2

Benefit: The protocol for the QuantiGene Plex Assay is 1,200 invariant regardless of the panel of genes that is measured. 6mm 6 mm 800

Unlike enzyme assays, no further optimization is required 6 mm 6 mm 400 when going from 1 panel to the next. The specificity of

Mean uorescence intensity uorescence Mean 0 QuantiGene Plex Assays allows closely related genes to be 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 measured simultaneously in the same well. Genes

Fold induction ofLiver mRNA FFPE sections FFPE liver sections—1 vs. 2 H&E-stained Unstained Section 1 Rifampin 30 section 1 section 2 1,600 Clofibrate Section 2 3-Methylcholanthrene Phenobarbital Ritonavir 1,200 Omeprazole 20 Phenytoin

ß-Napthoflavone 6mm 6 mm 800

6 mm 10 6 mm 400

Mean uorescence intensity uorescence Mean 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 old induction vs veicle control veicle vs induction old 1A1 1A2 1E1 2A6 2C6 2C9 2C18 2C19 2B6 2D6 2E1 3A4 3A5 Genes m CYP panel

Omeprazole dose response curve

CYP1A2 CYP2B6 CYP3A4 UGT1A4

10 4 2.5 3 EC50 2.008e–006 EC50 1.676e–005 EC50 8.585e–006 EC50 1.063e–005 8 2.0 3 2 6 1.5 2 4 1.0 1 1 old induction old induction old

2 induction old 0.5 induction old

0 0 0.0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 log log log log

UGT2B7 SLC22A1 SLC01B3 1.0 1.5 1.0

0.8 0.8 1.0 0.6 0.6

0.4 0.4 0.5 old induction old induction old

0.2 induction old 0.2

0.0 0.0 0.0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 log log log

8 Application 8: Classification Principal component analysis of 7 genes of melanoma subgroups using 4 Gene P value QuantiGene Plex gene signatures Metastatic melanoma Normal PCA BCL6 62 genes relevant to melanoma 2 PTEN were incorporated in a QuantiGene ARPC2 0 Plex Assay as a proof of concept to CXCL12 pca demonstrate the capability to discover –2 BRAF biomarkers. Tissue lysates were PCNA –4 prepared from frozen sections from CLEC3B –5 0 5 10 20 cases of metastatic melanoma ot melanoma lymp node pca it pigmented istiocytes 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.

9 Application 9: Stimulation of whole human blood CXCL10-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.

CXCL10 stimulation of whole blood for 6 hr and QuantiGene analysis of gene expression 7

0.3 µg CXCL10 6 1.0 µg CXCL10 1.0 µg CXCL10 Heat-inactivated 5

4 treated to untreated to treated 3

2 atio o CXCL10 1

0 CD86 CXCL5 IL1R1 IL8 IFNG CCL8 TNFP6 IL2RA CXCL10 IFIT3 Ci5

Specifications The QuantiGene Plex Assay can be used for both mRNA profiling and DNA copy number variation.

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 Cultured cells, bacteria, whole bood, PAXgene, Invitrogen™ Tempus™ samples, dried blood Compatible sample types 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

10 Off-the-shelf panels Custom panels We have also developed over 3,700 panels that cover Custom Quantigene Plex panels are available for any over 17,000 genes. These available panels cover a wide gene and any species. Custom panels ranging from 3- to range of host species that include: human, mouse, rat, 80-plex can be designed and shipped within 2 weeks. canine, monkey, porcine, soy, maize, and many others. Customers simply provide us with a gene list, RefSeq IDs, Any combination of existing genes can be combined or DNA sequences. 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]

Below are some examples of the genes included in the 80-plex panels The 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

Human Apoptosis 80-plex panel

CASP6 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 panel

NUSAP1 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 panel

IL1F6 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 panel

PYCARD 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

References 1. Beer DG et al. (2002) Gene expression profiles predict survival of patients with lung 5. Moeller T et al. Celsis—IVT − Transcript regulation of 18 ADME genes by prototypical adenocarcinoma. Nat Med. 8:816-824. inducers in human hepatocytes. Paper presented at: The 18th International Society 2. Ebsworth K (2007) Gene expression analysis by Quantigene Plex: validation for the Study of Xenobiotics. 18th North American Regional Meeting; 2012 Oct 14-18; and applications. Paper presented at: Planet xMAP USA; 2007 March 12-14; Dallas, TX. Laguna Hills, CA. 6. Leong SP et al. Paper presented at: The 4th International Symposium on Cancer 3. Hall JS et al. (2011) Exon-array profiling unlocks clinically and biologically relevant Metastasis and the Lymphovascular System: Basis for Rational Therapy; 2011 May gene signatures from formalin-fixed paraffin-embedded tumour samples. Br J Cancer 12-14; New York, NY. 6:971-981. 7. Witte A et al. Medarex—CXCL10 expression and biological activities in inflammatory 4. Wong S et al. New methodology for measuring mRNA levels along with monitoring CYP bowel disease. Paper presented at: The Digestive Disease Week Meeting; 2008 May activity in induction studies using human plateable cryopreserved hepatocytes. Paper 19-21; San Diego, CA. presented at: The 8th International Society for the Study of Xenobiotics; 2007 Oct 9-12; Sendai, Japan.

11 QuantiGene Plex panels—pathway guide

QuantiGene Plex pathway panels QuantiGene Plex pathway panels Human Mouse Rat Human Mouse Rat Apoptosis and autophagy Metabolism and endocrinology Apoptosis • • • Adipogenesis • • • Autophagy • • Fatty acid metabolism • • Unfolded response • • Glucose metabolism • • • Cancer signaling Diabetes • • • Angiogenesis • • • Glycosylation • • Angiogenic growth factors and angiogenesis inhibitors • • • Insulin signaling pathway • • • Breast cancer and estrogen receptor signaling • • • Mitochondria • • • Cancer pathway • • • Mitochondrial energy metabolism • • Cancer drug resistance and metabolism • • Hypertension • • • Cell cycle • • • Obesity • • • Epithelial to mesenchymal transition (EMT) • • Neuroscience Extracellular matrix and adhesion molecules • • • Neurogenesis and neural stem cell • • • Oncogenes and tumor suppressor genes • • Neuroscience ion channels and transporters • • • Tumor metastasis • • • Neurotransmitter receptors and regulators • • • Cardiology and bone biology Neurotrophin and receptors • • • Atherosclerosis • • Stem cells Osteogenesis • • • Embryonic stem cells • • Skeletal muscle—myogenesis and myopathy • • • Endothelial cell biology • • • Epigenetics Hematopoietic stem cells and hematopoiesis • • Epigenetic chromatin modification enzymes • • Mesenchymal stem cell • • Epigenetic chromatin remodeling factors • • Stem cell • • • Polycomb group (PcG) genes and DNA methylation • • Stem cell signaling • • Transcription factors • • Terminal differentiation markers • • Inflamation and immunology Toxicity and drug metabolism Cell surface markers • Cardiotoxicity • • • Chemokines and receptors • • • Drug metabolism • • • Cytokines • • • Drug metabolism phase I enzymes • • Cytoskeleton regulators • • Drug metabolism phase II enzymes • • Dendritic and antigen-presenting cell • • Drug transporters • • • HIV infection and host response • Hepatotoxicity • • • Inflammation • • Lipoprotein signaling and cholesterol metabolism • • • Inflammasomes • • • Nephrotoxicity • • • Inflammatory cytokines and receptors • • • Neurotoxicity • • • Inflammatory response and autoimmunity • • • Stress and toxicity • • • Innate and adaptive immune responses • • Toxicity • Interferon α and β response • • Housekeeping genes Interferon and receptor • Housekeeping genes • • • Interferon (IFN) and receptor mouse • Custom T cell and B cell activation • • • Any gene for any model organism • • • 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 • •

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