RT2 Profiler PCR Arrays: Pathway Analysis
Contents
1. 9 EL Ix O S 5 2 0 o Z 905 92 E 8 c 4 Cue Cue 8 N N Q SED d N a CN Oc D ose O Moe 5 adl rad z wre oe E 58 lt lt E L E OZ 0 dc v gt gt lt o mos amp 9 9 5 2 f O o 9 ZEO gt 30000000000000000 16600000000000009 300009009000000090 A 10000000000000000 20060000660006000 2606060000600000600 26000006060000000 R O 8 8838 M d e Qd E E On ED 0000000000000000 2 0000000000000000 7 0000000000000000 e0ede0e000000000 3 0000000000000000 e D 0 San HS N 0000000000000000 3 6666000000000000 0000000000000000 gt 20000000000000000 6000000000000000 5 lt o2 o d iz 0000000000000000 7 0000000000000000 L o 00000000060000 060006060060 0060000000000000 0 v co o 0000000000000000 2 0000000000000000 2 0000000000000000 0000000000000000 00090000 2E o0 qt eesseececseseseoo co 0000000000005 ooO00000000000000 2 e6060606060000000 00000000000600000 E o Qc E 0000000000000000 0000000000000000 C 6000000000000000 E 0000000000000000 5 6000000000000000 gt 2 ODS U 5 7 m 96000000000000000 0000000000000000 6000000000000000 X 6000000000000000 Z 6000 a 0000000000000000 000002. e C lt a N e lt gt 9 gt HS e P gt 2 lt QO 9 e A4 RT Profiler PCR Array is designed and parameters to insure the genes in your sample across a wide dynamic range are available in a number of easy to use formats for quick sample loading and data analysis list and receive your custom RT Profiler PCR Arrays in approximately 2 weeks Custom RT Profiler PCR Arrays employ a reproducibly recognized and quantified macaque drosophila or dog genomes up to c O E 9 o 2 O 2 gt E Q 2 v E lum c gt c um gt high throughput approach for profiling the expression of your genes of interest Choose from any gene in the human mouse rat rhesus 384 different genes Whether your interests are in biomarker discovery microarray followup drug development disease characterization or signal transduction mechanisms custom RT Profiler PCR Arrays enable focused expression E Performance Each assay in a custom W Flexibility Custom RT Profiler PCR Arrays are What are custom RT Profiler PCR analysis on your genes of interest Why custom RT Profiler PCR or 5 20 25 BEE S2 955 oF cet Q o P955 2 T O o O E2 d5 E FG a 20 E we cc D o o Ium Jg D T o0 c D o 0 gt
3. Signaling Pathway Oxidative Stress and Antioxidant Defense 53 Signaling Pathwa Wnt Signaling Pathway Cat no SAP st PCR Array 330231 PAXX 024Y 330231 PAXX 012Y 330231 PAXX O84Y 330231 PAXX 005Y 330231 PAXX 033Y 330231 PAXX 020Y 330231 PAXX 022Y 330231 PAXX 023Y 330231 PAXX 029Y 330231 PAXX 002Y 330231 PAXX 040Y 330231 PAXX 081Y 330231 PAXX O15Y 330231 PAXX 085Y 330231 PAXX O90Y 330231 PAXX 013Y 330231 PAXX 071Y 330231 PAXX O41Y 330231 PAXX O76Y 330231 PAXX 078Y 330231 PAXX O54Y 330231 PAXX 093Y 330231 PAXX 032Y 330231 PAXX O11Y 330231 PAXX 052Y 330231 PAXX O16Y 330231 PAXX O39Y 330231 PAXX 061Y 330231 PAXX 008Y 330231 PAXX 094Y 330231 PAXX 025Y 330231 PAXX 065Y 330231 PAXX 027Y 330231 PAXX O43Y www SABiosciences com XX species Y plate format RT Profiler PCR Arrays Pathway Analysis 01 2011 RT Profiler PCR Arrays What are RT Profiler PCR Arrays Profiler PCR Arrays are a highly reliable and sensitive gene expression profiling technology for analyzing focused panels of genes in signal transduction biological processes or disease research pathways using real time PCR Each cataloged RT Profiler PCR Array contains a list of the pathway focused genes as well as 5 housekeeping reference genes on the array In addition each array contains a panel of proprietary controls
4. 378 Chen A et al 2009 Endothelial cell migration and vascular endothelial growth factor expression are the result of loss of breast tissue polarity Cancer Research 69 6721 www SABiosciences com RT Profiler PCR Arrays Pathway Analysis 01 2011 RT Profiler PCR Array system How the RT Profiler PCR Array system works RT Profiler PCR Arrays are a complete system for pathway focused gene expression analysis From sample preparation to data analysis the RT Profiler PCR Array system includes four components that guarantee high quality reproducible and reliable gene expression data Integral to the performance of the RT Profiler PCR Array system is a proprietary set of control elements that enhance the reliability of your data and serve as a guarantee for performance over time These elements allow researchers to quickly assess the quality of their data by determining if samples were contaminated with genomic DNA gDNA the quality of the reverse transcription reaction and real time PCR efficiency Each component of the RT Profiler PCR Array system contributes to these quality control elements by incorporating an interlocked system for comprehensive monitoring of each step of the Process Cells tissues FFPE o blood and biofluids RT Profiler PCR Arrays 2 3 lt 2 Each pathway focused RT Profiler PCR Array includes 89 wet bench na verified RT qPCR Primer Assays in
5. RT Profiler PCR Array accessory RT First Strand Kit 12 samples Cat no 330401 Master Mix for RT Profiler PCR Arrays RT SYBR Green ROX qPCR Mastermix 2 arrays 96 well 12 arrays 96 well 24 arrays 96 well 4 arrays 384 well 2 arrays 96 well 12 arrays 96 well 24 arrays 96 well A arrays 384 well 2 arrays 96 well 12 arrays 96 well 24 arrays 96 well A arrays 384 well RT SYBR Green Fluor qPCR Mastermix RT SYBR Green qPCR Mastermix Cat no 330520 330522 Popular RT Profiler PCR Arrays for research Angiogenesis Apoptosis Autophagy Breast Cancer and Estrogen Receptor Signaling Cancer PathwayFinder Cell Cycle Chemokines and Receptors Diabetes DNA Damage Signaling Pathway Drug Metabolism EGF PDGF Signaling Pathway Embryonic Stem Cells Endothelial Cell Biology Epigenetic Chromatin Modification Enzymes Epithelial to Mesenchymal Transition Extracellular Matrix and Adhesion Molecules GPCR Signaling Pathway Growth Factors Heat Shock Proteins Hedgehog Signalin Pathway Hematopoietic Stem Cells amp Hematopoesis Hepatoxicity Hypoxia Signaling Pathway Inflammatory Cytokines and Receptors Innate and Adaptive Immune Response Interferon a p Response JAK STAT Signaling Pathway MAP Kinase Signaling Pathway Mitochondrial Energy Metabolism Nephrotoxicity
6. green chemistry yields earlier Cis for each dilution demonstrating better sensitivity than biological results Figure 4 Stress and Toxicity PathwayFinder RT Profiler PCR Array uncovered distinct gene expression profiles 5000 Ac tos TagMan chemistry 5005 TAS associated with liver toxicity caused by 3 PPARy agonists 125 128 RNA from HepG2 cells treated with three different E 119 glitazone agonists for type 2 diabetes mellitus was D J characterized and the results were compared to that of 30 a vehicle DMSO control The drug withdrawn due to 23 MS T M O 20 idiosyncratic liver toxicity Rezulin induces very different 6 changes in expression of stress related genes than two 10 safer drugs still on the market Avandia and Actos 0 I OI o gt Z lt 7 ABD A ze 2 8 2 gt Figure 5 Common Cytokine RT Profiler PCR Array 10 identified 23 up regulated and 6 down regulated genes 8 ILIO following PBMC stimulation Triplicate total RNA samples ia id TNF FNG CSF2 IL2 from human peripheral blood mononuclear cells either 10 HB ITA ili 6122 untreated or stimulated with 50 ng ml and 1 mg ml 10 gt e c 115 ME 3 ionomycin for 6 hours were characterized with the human 9 105 4 13 ere 2 PDGFA Common Cytokine RT Profiler PCR Array T
7. the genomic DNA reverse transcription efficiency and positive PCR control well data Make your pathway focused gene expression analysis quick and painless with the RT Profiler PCR Array system and the RT Profiler PCR Array Data Analysis Suite Simple Just upload your data and define your parameters B Convenient No downloading or installation required Publication ready output Export all results as free Excel files or png image files Excel based data analysis templates are available from our website Instructions Upload your data a simple Excel file format 2 Define your housekeeping genes and experimental groups 3 Choose an automatically generated data analysis result E Take a test run with pre loaded sample data set today www SABiosciences com pcrarraydataanalysis oh E Join our next live webinar entitled PCR Array Data Analysis Tutorial at www SABiosciences com seminarlist php 2 RT Profiler PCR Array Data Analysis Windows Internet Explorer a http perdataanalysis sabiosciences com pcr arrayanalysis php target volcanoplot on m Treated Group 1 w Control Control Group Group 1 vs Control Group 10010 P value Figure 14 The volcano plot indicates statistical significance of gene expression changes The x axis plots the log
8. to monitor genomic DNA contamination GDC as well as the first strand synthesis RTC and real time PCR efficiency PPC Isolate RNA from Why use Profiler PCR Arrays research samples of cells tissues FFPE and or blood Simplicity The simplicity of RT Profiler PCR Arrays makes routine expression profiling practical in any research laboratory with a real time instrument E Performance RT Profiler PCR Arrays have the sensitivity reproducibility specificity and reliability to accurately profile multiple genes simultaneously in 96 and 384 well plate 100 well disc and 96x96 S Convert total N RNA to cDNA chip formats Relevance RT Profiler PCR Arrays focus on profiling the genes relevant to the pathways or disease states important to your research Control Experimental Add cDNA to RT SYBR Green qPCR Mastermix RT Profiler PCR Array plate layout Aliquot mixture across RT Profiler 10 11 12 PCR Array A B C 30 31 Run in your D 42 43 real time PCR instrument E 001 amp 3 Emo 000 BX 8 8 Be FE BN 660669696 99999969 8 80 8 8 8 8 s 9 888 ERR NS 28060000002 on au nn m e E E et Q 1 001 Delta Rn SDM E WIY A4 f I P IP JT 7 j n M 4 1 E 002 Housekeeping Genomic
9. 00000000000 N QOOQOOOOO0O000000 7 0000000000000000 SOOO O 2 2 06006000000000000 0000000000000000 0000000000000009 co 0000000000000000 S 00004 N D lt 2 0909009099090909999999 OOOSSOSOOOHOOSOOS 20500000000000000 0000000000000000 SOOS o 9000000000000000 OOO00000000600000 3 6000000000000000 n 60600000006000000 8 ii D m Z ee Sw 42 0000000000000000 7 OOOOOOOOCOOOCOOO 84 0000000000000000 0000000000000000 9 c o 5 t 0000000000000000 QOOOO00000000000O0 6660000000000000 6000000000000000 7 D 2 DIL c 2 0000000000000000 660006000000060000 o 0000000000000000 o 000000000000000 L D a 2 a 6 OE 0000000000000000 M 60000000006000000 O000000000000000 6060000060000000 QOQOODOOO0000000 ay an 2 5 75 CD 0000000000000000 5 9000000000000000 0000000000000000 0000000000000000 0000990000000900 Soag 9000000000000000 0000000000000000 C 0000000000000000 0000000000000000 c 0000000000000000 me gt 2000000000000009 0000000000000009 0000000000000009 0000000000000009 600060008000056 9000000000000000 faovouwwOr aya 20 suaduvcor vYaA 20 lt 5 5 20 2260906 T eh Zzoa O 5 205 c co 2 On e gt lt N E D X 3 5L of LLI n
10. 2 of the fold differences while the y axis plots their p values based on student s test of your replicate raw C data The blue and red symbols outside the gray area conveniently have the same meaning as the scatter plot Symbols in the volcano plot above the dashed line readily identify fold differences at least as statistically significant as a threshold that can be defined www SABiosciences com RT Profiler PCR Arrays Pathway Analysis 01 2011 QIAGEN RNeasy Kits What are RNeasy Kits RNeasy Kits are a proven technology for rapid and convenient purification of high quality RNA Reproducible yields of intact RNA with high Agilent RIN RNA integrity number values are obtained enabling reliable results in downstream applications such as real time RT PCR Kits are available for cells and easy to lyse tissues as well as for more challenging samples such as fiber rich or fatty tissues fine needle aspirates and cryosections Why use RNeasy Kits Sample When purifying RNA it is important to use a method that maintains RNA integrity and removes Lyse homogenize contaminants Degradation of RNA makes reliable analysis of gene expression impossible while the presence of contaminants in the purified RNA can inhibit enzymes in downstream applications such as real time RT PCR and microarray analysis RNeasy Kits overcome Bind total RNA to these challenges thro
11. Cytokine Inflammasomes Inflammatory Cytokines and Receptors Inflammatory Response and Autoimmunity Interferon a B Response Interferon and Receptor JAK STAT Signaling Pathway Signaling Pathway T Cell Anergy amp Immune Tolerance T cell and B cell Activation BMP Signaling Pathway Th17 for Autoimmunity and Inflammation Th1 Th2 Th3 TNF Ligand and Receptor Toll Like Receptor Signaling Pathway Tumor Necrosis Factor TNF Ligand and Receptor Angiogenic Growth Factors amp Angiogenesis Inhibitors Atherosclerosis Chemokines and Receptors Common Cytokine Embryonic Stem Cells Endothelial Cell Biology Extracellular Matrix and Adhesion Molecules Glycosylation MAP Kinase Signaling Pathway Mesenchymal Stem Cell NF B Signaling Pathway Osteogenesis BMP Signaling Pathway TNF Ligand and Receptor Tumor Metastasis VEGF Signaling Wound Healing Alzheimer s Disease Apoptosis Autophagy Drug Transporters Embryonic Stem Cells GPCR Signaling PathwayFinder Heat Shock Proteins Hedgehog Signaling Pathway Huntington s Disease Hypoxia Signaling Pathway Mesenchymal Stem Cell Neurogenesis and Neural Stem Cell Neuroscience lon Channels and Transporters Neurotransmitter Receptors and Regulators Neurotrophin and Receptors Nitric Oxide Signaling Pathway Notch Signaling Pathway cAMP Ca2 Sign
12. LL z 2 0 Benefits of RT PreAMP system for FFPE samples 4 Quick and efficient High quality and high yield total RNA from FFPE samples with RNeasy FFPE Mini Kit PreAMP AAC Superior sensitivity RT PreAMP protocol significantly enhances Figure 12 Highly comparable gene expression fold change T results between FFPE preamplified and unamplified samples qRT PCR detection sensitivity for FFPE samples RNA extracted from FFPE spleen and intestine samples were extracted and converted to cDNA with and without preamplification All 4 cDNAs were analyzed on the Human Cancer PathwayFinder RT Profiler PCR Array The AAC RT PreAMP performance comparison and genes with raw C values lower than 33 in both unpreamplified spleen and intestine samples are presented Easy workflow Simple procedure and robust performance E Increased positive call rate from FFPE samples Increased detection of genes previously classified as absent Unbiased amplification of preamplified genes i Faithful conservation of biological changes Figure 13 Genes extracted from FFPE samples previously only ERT PreAMP RT CORT ERT PreAMP CORT ERT PreAMP classified as absent are now detectable after PreAMP preamplification RNA was extracted from FFPE spleen AO sample human kit and reverse transcribed to cDNA 3 using RT preamplification dark blue bars and without 3 PreAMP
13. N sample disruption products work and lung or RNeasy Lipid Tissue Mini Kit brain RNA was sled in a volume of 50 yl and concentration was The QlAshredder is a biopolymer shredding system in a spin column determined using a spectrophotometer format Cell lysate is applied to a QlAshredder spin column which is then briefly centrifuged to homogenize the lysate The TissueRuptor is a handheld device that provides simultaneous disruption and homogenization using TissueRuptor Disposable Probes which contain a blade that rotates at very high speeds As the probes are both disposable and transparent the risk of cross contamination is minimized and the sample disruption process can be visually monitored Use of disposable probes also saves time as there is no need to clean the same probe after disrupting each sample Tissuelyser instruments are bead mills that simultaneously disrupt and homogenize samples through high speed shaking with grinding beads in plastic tubes Using an adapter that holds several tubes the instruments disrupt multiple samples at the same time up to 12 samples with the TissueLyser LT and up to 48 or 192 samples with the TissueLyser Il Ordering Information Product Contents Cat no QlAshredder 50 For homogenization of cell lysates 79654 TissueRuptor For disruption of individual samples 9001271 TissueRuptor Disposable Probes 25 Disposable probes for use with the TissueRuptor 990890 Tissuelyse
14. RT Profil er PCR Arrays Pathway Anal 15 Gu ppoptos s piomarker The C 2 omplete PCR arra e p A a Conce research y 3 oe Cell Cycle dc Cytokines ECM adhesion techni chnical reference Neuroscience i Signo transduction evelopment Stem 9 drug ADME Sas A 4 Toxicology P up ap ap Sample ple amp Assay Technologies Table of contents RT Profiler PCR Arrays RT Profiler PCR Array system RT Profiler PCR Array performance RT PreAMP system for FFPE samples PCR array data analysis guide QIAGEN RNeasy Kits QIAGEN products for sample disruption QIAGEN products for RNA stabilization RT Profiler PCR Array quick index Compatible instruments QIAGEN Rotor Gene Q Rotor Gene 6000 Applied Biosystems ABI ABI 5700 7000 7300 ABI 7500 Standard ViiA 7 96 well block ABI 7500 FAST ViiA 7 FAST 96 well Block ABI 7900HT Standard 96 well Block 7700 ABI 7900HT FAST 96 well Block ABI 7900HT ViiA 7 384 well Block ABI StepOnePlus Bio Rad iCycler iQ 5 MyiQ MyiQ2 Chromo4 CFX96 Opticon 2 CFX384 Mx3000P Mx3005P Mx40009 Roche LightCycler 480 96 well Block LightCycler 480 384 well Block Eppendorf Mastercycler ep realplex 2 25 4 45 TaKaRa TP 800 Fluidigm BioMark Stratagene Plate 720 I gt gt 0 m gt o gt 217 gt
15. Reverse Positive sl So ATA sli NEAL genes DNA transcription PCR controls 7704 8 12 20 28 32 36 40 04 8 p 20 2 32 36 40 control controls Cycle Number ycle number Figure 1 Each well in an RT Profiler PCR Array measures the expression of a gene related to a pathway or disease state A typical 96 well format is shown This is also available in a 384 well plate 100 well disc and 96x96 chip format Data analysis Normal breast 9 v 9 8 p Value for fold change 105 105 10 10 102 10 1 Breast tumor 1 4 3 2 4 0 1 2 39 4 Fold change ratio log2 RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies R 0 97 1 Slope 0 99 12 M 86 genes 27 2 i m du g a 2 16 12 8 209 4 8 12 16 E 3 197 8 EA wae 10 16 RT PCR log FC Figure 2 Comparable biological results Gene expression analysis was compared between RT Profiler PCR Arrays SYBR Green based and the TaqMan platform Regression analysis of fold differences with data normalized against POLR2A demonstrate that both platforms yield similar 25 lll TaqMan 100 20 R 0 9998 E SYBR Green U 15 E 100 R 0 9999 10 4 6 8 Log copy number Figure 3 Sensitivity with RT SYBR Green versus TaqMan chemistry PCR amplicons detected using the same primer pair with or without TaqMan probes in either SYBR Green or TaqMan chemistry SYBR
16. aling PathwayFinder EGF PDGF Signaling Pathway G Protein Coupled Receptors GPCR Signaling PathwayFinder Heat Shock Proteins Hedgehog Signaling Pathway Insulin Signaling Pathway JAK STAT Signaling Pathway MAP Kinase Signaling Pathway mTOR Signaling Signaling Pathway Nuclear Receptors and Coregulators PI3K AKT Signaling Pathway Signal Transduction PathwayFinder BMP Signaling Pathway Transcription Factors Wnt Signaling Pathway Adipogenesis Dendritic and Antigen Presenting Cell Embryonic Stem Cells Hedgehog Signaling Pathway Hematopoietic Stem Cells and Hematopoiesis Homeobox HOX Genes Lipoprotein Signaling and Cholesterol Metabolism Mesenchymal Stem Cell Neurogenesis and Neural Stem Cell Neurotrophin amp Receptors Notch Signaling Pathway Osteogenesis Stem Cell Signaling T cell and B cell Activation Terminal Differentiation Marker TGFB BMP Signaling Pathway Wnt Signaling Pathway Cancer Drug Resistance and Metabolism Cancer PathwayFinder Cardiotoxicity Cell Cycle DNA Damage Signaling Pathway Drug Metabolism Drug Metabolism Phase Enzymes Drug Metabolism Phase Enzymes Drug Transporters GPCR Signaling PathwayFinder Hepatotoxicology Lipoprotein Signaling amp Cholesterol Metabolism Mitochondria Molecular Toxicology 38AHT Nephrotoxicity Oxidative Stress and Antioxidant Defe
17. ay focused PreAMP technology Figure 11 RT Profiler PCR Arrays detect RNA across a wide dynamic range Ten fold serial dilutions of human CHRNAS were characterized with the respective RT qPCR Primer Assay RT Profiler PCR Arrays are used and trusted by thousands of research scientists for pathway focused gene expression analysis Several factors including the RT qPCR Primer Assay design algorithm the proprietary control panel and the strict manufacturing and quality control procedures ensure the outstanding performance and reliability of our RT Profiler PCR Arrays Each RT Profiler PCR Array and every RT qPCR Primer Assay is wet bench verified to guarantee their performance with results demonstrating several performance parameters illustrated here Distinct specificity The complete RT Profiler PCR Array system with high quality input RNA is guaranteed to yield single bands without primer dimers or other secondary products The proprietary primer design algorithm incorporates more than 10 thermodynamic and sequence alignment criteria and our wet bench verification provides confidence that every real time qPCR assay accurately represents the expression of the queried gene Over 20 000 gene specific RT qPCR Primer Assays have been designed and shipped to satisfied customers High sensitivity and wide dynamic range A key benefit of using pathway focused RT Profiler PCR Arrays for gene expression analysis is that gene
18. cluding 5 housekeeping genes and a proprietary control panel RT SYBR Green qPCR Mastermixes A unique formulation of buffers that co evolved with the primer design P v algorithm provides high amplification efficiencies Available with E 2 2 Buffer GE 2 reference dyes ROX Fluorescein or without 2s RT enzyme lt RT First Strand Kit E 2 An external RNA control detected by the RT Profiler PCR Array tests 2 8 Random 5 5 g hexamers 5 the quality of input RNA It also features a proprietary genomic DNA 5 PR o External RNA elimination buffer essential for eliminating residual gDNA ensuring 5 control RTC specific detection of mRNA dNTPs m Free data analysis software Fi d cDNA The power of the RT Profiler PCR Array to assess the expression of a x pathway focused set of genes over a wide range of detection yields E an abundance of data With our free RT Profiler PCR Array data 2 E a gt d nne analysis tool go from raw values to fold change results displayed Si dii S dNTPs in a variety of formats scatter plots volcano plots clustergram a 5 matter of minutes ONCE 2 sie DNA e B polymerase Z binding x j 2 Elongation 519 SYBR Green E 5 binding e Detection RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies TRizol Mg Addition treatment verified using a set of rigorous
19. i O E I uoo Q Rca oe 220 Sie iQ oc oe 00000000 EROOOOOO0O00 200000000 200000000 Sm 0000000 oooooooo 00000000 00000000 00000000 E sas a2 5 90000000 s 200000000 200000000 200000000 200000000 lt lt ES a e 0000000 090000000 gt 00000000 7 00000000 00000000 gt E c 5 906000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 5 00000000 5 00000000 E D D o E o 006000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 90000000 00000000 vo vo N N gt 00000000 00000000 090000000 00000000 00000000 QO o 99000000 5 090000000 5 00000000 00000000 00000000 00000000 090000000 90000000 00000000 00000000 lt e00000000 00000000 00000000 00000000 00000000 lt or lt c OQ OQ 1 uw OT MECP2 Tg MECP2 null 8 6 5 ctivated Repressed 2 gt o 5 2 A Gene 6 Figure 8 Gene expression changes in hypothalamus of MECP2 mouse models Validation of expression changes for 66 genes by qPCR analysis Gene expression levels from microarray analyses were validated in four MECP2 Tg males and four Mecp2 null males Data is plotted as relative up regu
20. lation light blue or down regulation dark blue over wild type P lt 0 05 t test Each column represents a single gene and represents data from four samples for each genotype 96 well platet or 100 well disc custom RT Profiler PCR Arrays Custom RT Profiler PCR Array Rotor Gene Q format Format Number of arrays minimum 9 gengs 12 sampies piate 100 well disc format 12 genes 8 samples plate 12 16 genes 6 samples plate gg e 24 genes 4 samples plate 32 genes samples plate 2 2 48 genes 2 samples plate 96 genes 1 sample plate All formats Per additional 12 arrays amp E eoo g 9 an o 384 well custom RT Profiler PCR Arrays 60000000099 12 genes 8 sample plate Format Number of arrays minimum Pr aie N 8 genes 48 samples plate ES NY 12 genes 32 samples plate 6 z 16 genes 24 samples plate 8 24 genes 16 samples plate 32 genes 12 samples plate 3 o 48 genes 8 samples plate Xe 3 64 genes 6 samples plate REN y 96 genes 4 samples plate EC 128 genes 3 sample plate 96 genes 1 sample plate 192 genes 2 samples plate 24 384 genes 1 sample plate All formats Per additional arrays RT Profiler PCR Array Modified RT Profiler PCR Arrays Number of arrays minimum accessories Pack size Catalog Add up to 4 genes to a cataloged RT PreAMP cDNA 12 samples 330451 RT Profiler PCR Array 96 well 100 well 24 Sy
21. light blue bars Results of the Human Cancer PathwayFinder RT Profiler PCR Array showed 55 of 2 unpreamplified genes were virtually undetectable with no genes in the 10 20 C range Preamplified genes with 2 C values gt 30 shift into the reliably quantitative range 15 C 10 30 Ss A5 Raw C Oc Q I Information Product Cat no RT PreAMP cDNA Synthesis Kit 330451 RT PreAMP Pathway Primer Mixes for all RT Profiler PCR Arrays 330241 RT2 PreAMP Primer Mixes for custom RT Profiler PCR Arrays 330141 RT Profiler PCR Arrays Varies RT SYBR Green qPCR Mastermixes Varies RNeasy FFPE Kit 50 73504 RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies RT Profiler PCR Array data analysis Free web based RT Profiler PCR Array data analysis software This integrated web based software package for the RT Profiler PCR Array system automatically performs all AAC based fold change calculations from your uploaded raw threshold cycle data Simply providing the array s catalog number annotates the results to the correct gene list The web portal delivers results not only in a tabular format but also in scatter volcano cluster gram and multi group plots Perform any pair wise comparison between groups of experimental replicates by defining your own fold change and statistical significance thresholds or compare all of the groups side by side The web portal also helps to correctly interpret
22. lls in 96 well format 74182 QIAzol Lysis Reagent 200 ml For lysis of fatty and standard tissues before RNA isolation 79306 Automatable on the GlAcube Find out more at www qiagen com goto QlAcube RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies QIAGEN products for sample disruption What is sample disruption Effective disruption and homogenization of a biological sample is an absolute requirement for all RNA purification procedures Disruption releases the RNA contained in a sample while homogenization reduces sample viscosity to facilitate subsequent RNA purification 400 Why use QIAGEN sample disruption products 300 QIAGEN provides a range of technologies for disruption and homogenization from QlAshredder spin columns for fast and simple homogenization of cell lysates to TissueRuptor and Tissuelyser systems 199 for mechanical disruption and homogenization of tougher tissue samples RNA concentration ng pl O at a range of throughputs TissueRuptor and TissueLyser systems deliver Skin Heart Lung Brain fast and effective disruption and replace tedious and time consuming Figure 16 Effective tissue disruption Various rat tissues methods such as disruption using mortar and pestle were disrupted using the TissueLyser LT or Tissuelyser Il RNA was purified from 20 mg samples on the QlAcube e e using the RNeasy Fibrous Tissue Mini Kit skin heart How do QIAGE
23. matopoiesis Homeobox HOX Genes Mesenchymal Stem Cell Stem Cell T cell and B cell Activation Th1 Th2 Th3 Angiogenesis Apoptosis Breast Cancer and Estrogen Receptor Signaling Cancer Drug Resistance and Metabolism Cancer PathwayFinder Cell Cycle DNA Damage Signaling Pathway EGF PDGF Signaling Pathway Epithelial to Mesenchymal Transition EMT MAP Kinase Signaling Pathway p53 Signaling Pathway PI3K AKT Signaling Pathway Protein Phosphatases BMP Signaling Pathway Tumor Metastasis Tumor Suppressor Genes WNT Signaling Pathway Apoptosis Autophagy Cancer PathwayFinder Cell Cycle DNA Damage Signaling Pathway DNA Repair Epithelial to senchymal Transition EMT MAP Kinase Signaling Pathway mTOR Signaling Neurogenesis and Neural Stem Cell NF B Signaling Pathway p53 Signaling Pathway PI3K AKT Signaling Pathway Protein Phosphatases Signal Transduction PathwayFinder Transcription Factors Ubiquitination Pathway RT Profiler PCR Array free data analysis tool see page 10 htt www SABiosciences com sabiosciences com crarraydataanalysis RT Profiler PCR Arrays Pathway Analysis 01 2011 Inflammation Research ECM Adhesion Research Neuroscience Research Signal Transduction Research x Stem Cell Research Toxicology Drug ADME Research s Chemokines amp Receptors Common
24. nse Stress and Toxicity PathwayFinder RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies www SABiosciences com www qiagen com USA Orders 1 301 503 3187 Fax 1 301 465 9859 Technical 1 888 503 3187 Australia Orders 1 800 234 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 00800 22448000 Belgium Orders 0800 79612 Fax 0800 79611 Technical 00800 22448000 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 362 7737 China Orders 86 2 1 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 00800 22448000 Finland Orders 0800 914416 Fax 0800 914415 Technical 00800 22448000 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 00800 22448000 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 00800 22448000 Ireland Orders 1 800 555 049 Fax 1 800 555 048 Technical 00800 22448000 Italy Orders 800 789 544 Fax 02 334304 826 Technical 00800 22448000 Japan Orders 03 5547 0811 Fax 03 5547 0818 Technical 03 5547 081 1 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 00800 22448000 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 436 Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 00800 22448000 Nor
25. nthesis Kit ac RT PreAMP Pathway 12samples 330241 Add up to 4 genes to a cataloged Primer Mixes RT Profiler PCR Array 384 well pathway focused Chahrour M et al 2008 MeCP2 A key contributor to neurological disease activates and represses transcription Science 320 1224 t Also available in 96x96 Fluidigm BioMark format RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies RT Profiler PCR Array performance CXCRI CXCR2 2 i o A 0 1 Signal d RFU dT o 40 50 60 70 80 90 99 Tm C Figure 9 RT Profiler PCR Arrays amplify a single gene specific product in every reaction Universal total RNA was characterized for four chemokine and chemokine receptors using RT qPCR Primer Assays followed by a dissociation melt curve analysis RT Profiler PCR Arrays specifically detect individual genes despite the expression of related gene family members in the same RNA sample n 100 80 60 40 20 positive call 100 50 25 10 Input RNA ng Figure 10 RT Profiler PCR Arrays detect as little as 1 ng of RNA Different amounts of universal total RNA were characterized using the Human Inflammatory Cytokines and Receptors RT Profiler PCR Array PAHS 011 with or without PreAMP The percentage of detectable genes was calculated for each RNA amount with 1 ng RNA analysis enabled with the new pathw
26. ot specifically marked as such are not to be considered unprotected by law 2011 QIAGEN all rights reserved RT Profiler PCR Arrays Pathway Analysis 01 2011 Sample amp Assay Technologies RT Profiler PCR Array quick index Research area Apoptosis Research Biomarker Research Cancer Research Cell Cycle Research RT Profiler PCR Array listing For catalog numbers see page 2 or visit www SABiosciences com Arraylist php Try RT Profiler PCR Arrays Risk free starter pack offer details www SABiosciences com PCRguide php Apoptosis Autophagy Cancer PathwayFinder Cell Cycle DNA Damage Signaling Pathway DNA Repair Endothelial Cell Biology Heat Shock Proteins Signaling Pathway Oxidative Stress and Antioxidant Defense p53 Signaling Pathway PI3K AKT Signaling Pathway Stress and Toxicity PathwayFinder TNF Ligand and Receptor Tumor Suppressor Genes Ubiquitination Pathway Unfolded Protein Response Alzheimer s Disease Angiogenesis Breast Cancer and Estrogen Receptor Signaling Cancer PathwayFinder Cell Surface Markers Dendritic and Antigen Presenting Cell Epigenetic Chromatin Modification Enzymes Epigenetic Chromatin Remodeling Factors Epithelial to esenchymal Transition EMT Extracellular Matrix and Adhesion Molecules Glucose Metabolism Hematopoietic Stem Cells and He
27. r LT For disruption of up to 12 samples 85600 Tissuelyser LT Adapter 12 tube For purification of RNA from cells in 96 well format 69980 Tissuelyser ll For disruption of up to 48 or 192 samples 85300 Tissuelyser Adapter Set 2 x 24 Adaptor set for use with the Tissuelyser Il holds 48 tubes 69982 www SABiosciences com RT Profiler PCR Arrays Pathway Analysis 01 2011 QIAGEN products for RNA stabilization What is RNA stabilization Once a biological sample is harvested its RNA becomes extremely 100 Allprotect PBS O unstable The RNA is degraded by RNases and gene induction or downregulation triggered by sample manipulation will also occur Immediate stabilization of cellular RNA to preserve mRNA levels is KR O critical for accurate gene expression analysis N O Fold change in transcript level RNA stabilization is usually achieved by rapidly freezing samples in o lt 2 liquid nitrogen or on dry ice However the use of such chemicals is OS o Vv hazardous and care should be taken to avoid thawing of samples prior to sample disruption and RNA purification Why use QIAGEN RNA stabilization products QIAGEN provides a broad range of reagents for convenient stabilization Fold change in transcript level 0 1 of RNA in cells tissues blood and saliva at room temperature The use of hazardous liquid nitrogen or dry ice to freeze samples is avoided om Sample
28. s are simply submerged in the reagents to immediately preserve e the gene expression profile and can then be conveniently handled 5 lt 2 NS S qv vw qv and transported at ambient temperature prior to RNA purification For f lizati lab f Figure 17 Effective RNA stabilization Rat tissues were stored urther convenience stabilization reagents are also available as part o at 25 C for 2 24 hours in Allprotect Reagent or PBS prior to GIAGCEN kits tor PNA purification real time RT PCR analysis P Rat lung tissue was analyzed P for c fos expression El Rat intestine tissue was analyzed for Madh7 expression Transcript levels relative to those in liquid nitrogen stabilized tissues were calculated Changes in transcript levels were prevented by Allprotect Reagent Ordering Information Product Contents Cat no Allprotect Tissue Reagent 100 ml For stabilization of RNA DNA amp protein in tissues 76405 AllPrep DNA RNA Mini Kit 50 For simultaneous purification of DNA and RNA 80204 RNA ater RNA Stabilization Reagent 250 ml For stabilization of RNA in tissues 76106 RNeasy Protect Mini Kit 50 For stabilization of RNA in tissues and RNA purification 74124 RNAprotect Cell Reagent 250 ml For stabilization of RNA in cells 169206 RNAprotect Animal Blood Tubes 50 x 100 pl For collection of 100 pl animal blood with RNA 76544 stabilization RNeasy Protect Animal Blood Kit 50 For purifica
29. s that are over expressed can be measured as reliably as those that are under expressed The complete RT Profiler PCR Array system yields gt 85 positive call with 25 ng 5 ug RNA or gt 90 with as little as 1 ng PreAMP RNA with the RT PreAMP System The 8 log wide dynamic range provided by real time PCR is unparalleled when comparing a pathway focused gene panel of varying expression levels across a variety of samples LLL 0 2 A 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cyde number 9 995 C e O Fluorescence f O RT Profiler PCR Arrays Pathway Analysis 01 2011 www SABiosciences com RT PreAMP System for FFPE samples Gene expression analysis from FFPE samples An innovative solution enabling the accurate qRT PCR analysis of formalin fixed paraffin embedded FFPE samples The RT PreAMP technology utilizes multiplex tandem PCR to preamplify gene specific cDNA with minimal bias This kit is intended for preamplification of first strand cDNA from fragmented total RNA from FFPE samples for gene expression analysis with RT Profiler PCR Arrays The combination of a simplified RNA extraction and a high fidelity amplification process maximizes recovery of RNA RT Profiler PCR Arrays facilitate easy and reliable expression analysis of genes associated with J a biological pathway or a disease state from FFPE samples 4 uU oe P e E
30. tion of RNA from blood collected in VIDA RNAprotect Animal Blood Tubes miRNeasy Protect Animal Blood Kit 50 For purification of RNA including miRNA from blood 217304 collected in RNAprotect Animal Blood Tubes For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents Trademarks QIAGEN QlAcube GlAxcel QIAzol AllPrep RNAprotect RNeasy Rotor Gene TissueRuptor QIAGEN Group SYBR Molecular Probes Inc Roche LightCycler TaqMan Roche Group Applied Biosystems ROX StepOnePlus Applera Corporation or its subsidiaries Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P Mx3000P Mx4000 Stratagene Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ Bio Rad Laboratories Inc Fluidigm BioMark Fluidigm Corp Agilent Agilent Technologies Excel Microsoft Corporation TRIzol Molecular Research Center Inc Actos Takeda Pharmaceutical Company Limited Avandia GlaxoSmithKline Rezulin Parke Davis Pharmaceuticals Ltd Registered names trademarks etc used in this document even when n
31. ugh the combination of a specialized lysis buffer and silica membrane RNeasy membrane technology How do RNeasy Kits work Wash Biological samples are first lysed in a lysis buffer that contains a guanidine salt which fully denatures RNases to prevent RNA degradation RNA is then specifically bound to a silica membrane either in an RNeasy spin column or the well of an RNeasy 96 plate Other cellular Elute in small volume material is efficiently washed away using a series of wash buffers before pure intact RNA is cach 4 em aut em eluted in RNase free water Ready to use RNA 400 300 gt 200 a 100 0 5 10 min qo Td 9 Ss Ss se Figure 15 Highly intact RNA RNA was purified from Jurkat cells using the RNeasy Mini Kit The purified RNA was analyzed on the EVQIAxcel system ratio of 285 to 185 rRNA 1 55 and BlAgilent 2100 Bioanalyzer ratio of 28S to 185 rRNA 1 7 A high RNA integrity number RIN of 9 6 was obtained indicating highly intact RNA Ordering Information Product Contents Cat no RNeasy Mini Kit 50 For purification of RNA from cells amp easy to lyse tissues 74104 RNase Free DNase Set 50 For DNase digestion RNA purification 79254 RNeasy Fibrous Tissue Mini Kit 50 For purification of RNA from fiber rich tissues 74704 RNeasy Plus Universal Mini Kit 50 For purification of RNA from alll tissue types 73404 RNeasy 96 Kit 12 For purification of RNA from ce
32. way Orders 800 18859 Fax 800 18817 Technical 00800 22448000 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Sweden Orders 20 790282 Fax 020 790582 Technical 00800 22448000 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 00800 22448000 UK Orders 01293 422 91 Fax 01293 422 922 Technical 00800 2244800 QIAGEN T Sample amp Assay Technologies
33. wenty three 5 104 e TNFSFI1 IL17 cytokine genes are up regulated gt 5 fold p lt 0 0005 Je ES TNFRSPSF11B including interleukins colony stimulating factors TNF 10 IFNAS 4 MES 14 ligands after 6 hours of stimulation Six interleukin TNF ligand genes are down regulated under the same gt conditions 3 5 7 9 11 13 15 17 Figure 6 Relative fold change between disorganized and Fold difference organized colonies using the Angiogenesis RT Profiler PCR Array RNA isolated from unorganized T4 2 cells treated with a control antibody IgG or reverted to an 50 organized colony by blocking EGFR signaling mAb225 was reverse transcribed and relative gene expression ed 0 data was obtained using the human Angiogenesis Profiler PCR Array The expression profile of 84 genes t E relevant to angiogenesis as well as 5 housekeeping genes 9 100 were assayed Fold change calculations were done using 2 SABiosciences data analysis software which automatically 150 calculates fold change gene expression between the o treated and control groups 200 ognn ogoezQ o zozahoS9Ss8o 275mi52z3 amp EEDE EPEE EEEIEE EE POEP CT Arikawa E et al 2008 Cross platform comparison of SYBR Green real time PCR with TaqMan PCR microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control MAQC study BMC Genomics 9
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