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Quantigene Assays for Analysis of Gene Expression Or Copy Number Variation

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Quantigene Assays for Analysis of Gene Expression Or Copy Number Variation QuantiGene assays for analysis of gene expression or copy number variation Quantitate up to 80 genes 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 gene expression 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 CXCL10 10 –4 1 2 Normalized to skin broblast 0 Ratio of 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 TNFalP6 IL2RA CXCL10 IFIT3 Cig5 Mean uorescence intensity 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 –9 S100A8 BCL6 1.28 x 10 Rifampin HCC70 HCC3153 MDAMB468 SUM1314 BT20 H5578T BT549 MDAMB157 MDAMB231 MDAM453 LY2 SUM185PE T47D HCC202 BT474 ZR751 MDAMB361 AU565 SKBR3 KRT5 30 Clofibrate CTA-55I10.1 2 –8 TRIM29 3-Methylcholanthrene PTEN 1.03 x 10 S100A2 Phenobarbital FAT2 Ritonavir CLCA2 –8 Omeprazole ARPC2 2.17 x 10 DSG3 20 DSC3 Phenytoin 0 TP63 ß-Napthoflavone –7 EN1 CSTA CXCL12 1.37 x 10 DENND2C FOXC1 USH1C pca2$x[.2] –7 TSPAN8 10 –2 BRAF 4.18 x 10 GABRP KIF12 HGD M17 AC105391.3 –7 ERBB2 PROM1 PCNA 6.33 x 10 DDAH1 CA12 SPINK1 0 –4 –6 EPS8L3 control vehicle vs.
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