pcr array 2013

Sample & Assay Technologies

Pathway-powered PCR arrays

Advanced Real-Time PCR Technology to Empower Your Pathway Analysis

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Questions? Comments? or Suggestions?Ask now or contact Technical Support

Telephone: 888-503-3187 Email: [email protected]

Webinar related questions: [email protected]


Pathway-focused solution for expression analysis- Complete experimental systems: sample prep through data analysis

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SABiosciences.com


Pathway-focused solution for expression analysis- Complete experimental systems: sample prep through data analysis

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SABiosciences.com

http://sabiosciences.com/NGS.php

GeneRead NGS System

Qiagen.com


Pathway-focused expression analysis- Complete Experimental Systems: Sample Prep through Data Analysis

Genes (mRNA)

microRNA (miRNA)

DNA Methylation

Cancer Mutation

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Gene expression regulates biology

All require molecular signaling for action

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Biological markers define processes

Cell cycle Angiogenesis

p53MDM2CyclinsCDKs

VEGFbFGF

ANGPTPDGF

IL8TLRIFNγTNFβIn

divi

dual

pa

rtic

ipan

ts

Inflammation

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Pat

hway

Cell cycle Angiogenesis Inflammation

Complete biological story- Built on pathway / network analysis

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Differential gene expression

Q: How to assess the expression of different mRNAs in a sample involved in a process and compare it across multiple conditions?

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A:


Cancer and Apoptosis Cytokines & Inflammation Develop ment & Stem Cells

Apoptosis Inflammatory Cytokines Stem Cells

Cell Cycle Th17 for Inflammation WNT Signaling / Notch Signaling

Human miRNA Array (NEW!) Common Cytokines / Chemokines Terminal Differentiation Markers

Breast Cancer & Estrogen Receptor Inflammasomes TGFβ / BMP Signaling

Tumor Metastasis NF-kB Signaling Pathway Endothelial Cell Biology

Epithelial-to-Mesenchymal Transition Th1-Th2-Th3 Osteogenesis

Angiogenesis TNF Ligands Growth Factors

Cancer Drug Resistance Toll-like Receptors ECM & Adhesion

Signal Transduction Toxicology & Drug Metabolism Neur oscience

Signal Transduction PathwayFinder Drug Metabolism / Drug Transporters Neuroscience Ion Channels

NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors

Jak / Stat Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins & Receptors

DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell

Insulin Signaling Stress & Toxicity Parkinson’s Disease

MAP Kinase Signaling Other Diseases Custom PCR Arrays (H/M/R/Q/D/F/P/B)

cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate

p53 Signaling Diabetes 100-Well Disc, 96x96 Chip

PCR Arrays for all biomedical researchers

Over 140 Pathway- powered PCR arrays available

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All pathways @ www.SABiosciences.com

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Topics will be covered

1. Introduction to PCR Arrays

2. How PCR Arrays Work

3. Performance of PCR Arrays

4. Applications for PCR Arrays

5. What’s next after PCR Arrays

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PCR Array – plate format

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384-Well Human Inflammatory Cytokines & Receptors RT2 Profil er PCR Array

4x96

370

Sample & Assay Technologies- 13 -

� Amplification plot

PCR efficiency

� Melting/dissociation curve

Single peak

Primer assays- Laboratory Verification Data

Note: If you are designing your own primers STOP


qPCR primer assay & custom PCR array- Species Coverage

Wet-bench validated assays for all genes in:• Human: H/HS

• Mouse: M/MM

• Rat: R/RN

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Cow (Bos taurus) Chicken (Gallus gallus)

Horse (Equus ferus caballus) Zebrafish (Danio rerio)

Dog (Canis lupus familiaris) Pig (Sus scrofa)

Fruit Fly (Drosophila melanogaster) Rabbit (Oryctolagus cuniculus)

Chinese Hamster Ovary (CHO, Cricetulus griseus) cell

Rhesus macaque (Macaca mulatta)

Custom PCR Array- expandable to the following species


Biologically relevant gene content

• Not simply biochemical pathways or kinase cascades

• Published association with the biological or disease

pathway gathered from overlapping sources, including:

• Multiple Publicly Accessible Databases

• Text Mining Relevant Literature Genes in Signaling Pathways

Genes with High Relevance

Technically relevant gene content

• Use genes that are regulated at the mRNA level

• Specific feedback from thought leaders

How genes on PCR arrays are selected ?

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How genes on PCR arrays are selected- Published sources

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http://gncpro.sabiosciences.com/gncpro/gncpro.php


Relevant pathway content on PCR arrays

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Contents will be covered

1. Introduction to PCR Arrays

2. How PCR Arrays work




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Principles of qRT-PCR: overview

• Real-Time PCR

• Amplify and simultaneously quantify target DNA

• RT2 PCR (Reverse transcription teal-time PCR)

• Amplify and simultaneously quantify mRNA

• Ct Values: Threshold Cycle


Anatomy of a RT2 PCR Array

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• 84 Pathway-Specific Genes of Interest

• 5 Housekeeping Genes

• Genomic DNA Contamination Control

• Reverse Transcription Controls (RTC) n=3

• Positive PCR Controls (PPC) n=3

B2M, HPRT, RPL13A, GAPDH, HGDC

Sample & Assay Technologies- 21 -

Stimulate Cells Isolate RNA

RT-PCRData Analysis

How RT2 Profiler PCR Arrays work- Experimental workflow

Convert Total RNA to cDNA

Treat cells, for example non-cancer or cancer cells Isolate RNA (Rneasy Kits)

RNase-Free DNase TreatmentGenomic DNA Removal stepReverse Transcription step

5mins + 20 mins

+ SYBR Green Master MixReal-time PCR Detection

2hrs

Upload raw data (Ct) and analyze data15 mins

control sample


Interpretation of Ct values- Fold change calculations: PCR Array results / data

Raw Ct Value: Relative expression level of that gene in the sample1

3

2

Up or down-regulation= ∆∆Ct per Gene = ∆Ct

Condition 2 - ∆Ct Condition 1

Fold change results per gene = 2-∆∆Ct

Data normalization = ∆Ct per Gene = Ct Gene X - Ct HKGs (Avg)

Ex. My gene is present 4X more in the treated sample than in control sample.Now not only gene, but GENES in a Pathway (some up, some down)

�� Free complete & easy data analysis with web-based software

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• Gene content for each pathway is pre-loaded• Custom Arrays: Gene List is all you need

• From raw C t to fold change results in multiple analysis formats

Volcano Plot Scatter Plot Clustergram 3-D Histogram

RT2 Profiler PCR Array data analysis- FREE complete & easy analysis with web/excel-based software

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• Cultured cellsQIAGEN RNeasy Kits with On-Column DNase Treatment

• TissuesQIAZOL followed by RNeasy Kits with On-Column DNase Treatment

• Blood samplesQIAGEN PaxGene Blood RNA kit

• Small samplesQIAGEN RNeasy Micro Kit & RT2 PreAMP system

• Fine needle biopsy (FNB) samples • Laser captured microdissection (LCM) samples• Stem cell clusters or embryoid bodies • Fluorescence-Activated Cell Sorter (FACS) generated cells

• FFPE (Formalin-fixed Paraffin-embedded) samplesQIAGEN RNeasy FFPE Kit & RT2 PreAMP system

PCR Arrays compatibility- All sample types

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• 96-Well Blocks: 7000, 7300, 7500, 7700, ViiA 7

• FAST 96-Well Blocks: 7500, 7900HT, Step One Plus, ViiA 7

• FAST 384-Well Block: 7900HT, ViiA 7

• iCycler, MyiQ, MyiQ2 , iQ5, CFX96, CFX384• Opticon, Opticon 2 , Chromo 4

• Mastercycler ep realplex 2/2S/4/4S • BioMark

• LightCycler 480

• Mx3000p, Mx3005p, Mx4000p • TP-800

• Rotor-Gene Q, Rotor-Gene 6000

� No real time PCR machine? PCR Array Service from SABioNew Instrument Set-up protocol available

PCR Array compatibility- All RT-PCR instruments, 96-, 100-, 384-, and 96x96 well formats

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1. Introduction to the PCR Arrays


3. Performance of PCR Arrays– All individual PCR Primer Assays are wet-bench validated for:

• Sensitivity (25 ng – 5 µg)– Enhanced Sensitivity with PreAMP Technology (1 ng)

• Specificity• Reproducibility• Amplification Efficiency• Wide Dynamic Range


5. miRNA PCR Arrays & qBiomarker DNA Mutation PCR Arra ys & EpiTect DNA Methylation PCR Arrays

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Human Inflammatory Cytokines & Receptors PCR Array tested with Universal Reference RNA

Conclusion: See gene expression as rare as UN-induced cytokines.

• Human Inflammatory Cytokines & Receptors Array used to analyze un-induced cells. Genes represented at very low copy numbers.

• 25 ng - 1 µg total RNA per 96-well plate*

• 99% of genes detectable with > 500 ng

• > 80% of genes detectable with 25 ng

• Very sensitive assay!

High sensitivity- 25 ng to 5 ug of RNA

*RT2 PreAMP: 1 ng RNA

*

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Reproducibility Among Different Instruments

Human Drug Metabolism PCR Array in Universal Reference RNA

Conclusion: Same raw threshold cycle data can be obtained for the same samples even from different end users at different times or using different instruments .

Reproducibility Among Different Users

High reproducibility- With different users and different instruments

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Conclusion: Accurate and reliable ∆∆Ct results are guaranteed.

Uniform high PCR amplification efficiency

Cycle Gene 1: Your Design (AE=100%)

Gene 1: Friends Design (AE=85%)

0 1 1

1 2 1.85

2 4 3.42

..

35 3.4x1010 2.2 x109

∆ 15x Difference

Why this matters?

Does NOT reflect biology….rather, a technical issue� But you would have reported this as a biological finding

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Conclusion : Wide dynamic range to accommodate varying levels of initial gene expression.

Human Acetylcholine Receptor αααα5

Wide dynamic range

Measure genes that are overexpressed as well as tho se genes that are underexpressed

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Technical• Not necessary Save time & money• Reproducibility of the PCR Arrays is very high• Results demonstrate that what you are seeing is a result of

biology, not technique• RTC & PPC show technical reproducibility on each plate, and

comparable across plates.

Biological• Needed to verify the results are a result of biology • At least 3 replicates per condition for statistical analysis

-> p values

Replicates: technical & biological

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1. Introduction to the PCR Arrays






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• Revealed up- and down- regulated genes in breast cancer.

Application example 1- Tumor analysis

Many questions raised

1. What if knockdown all ↑genes & Induce all ↓ genes?� Revert to Normal?

2. Why TIMP3 and MMP3 up together?� And why at different rates?

3. Correlations between MMP3 & MMP9?

Spend your time Analyzing & Thinking

About Your Data!

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� Uncovered distinct gene expression profiles associated with liver toxicity caused by 3 PPARγ agonists (Actos, Avandia, and Rezulin).

Application example 2- Drug development / toxicity studies

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• Choose any gene from the human, mouse, rat, drosoph ila, rhesus macaque, dog, or pig genomes

Custom 100-well discs also available

12 Genes : 8 Samples


96-well 384-well



96 Genes : 1 Sample

• Analyze the genes most important to your research

• Available for any real-time PCR instrument.

• Wet-bench validated design & performance.

• 4 to 6 week turn around time first order (1 - 2 weeks in U.S.)

• 1 week turn around on re-order (2 - 3 days in U.S.)

• Same data analysis as cataloged arrays

# of samples x 4

Create your own PCR arrays- Custom formats

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What’s next - after PCR Arrays

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� Validate results with more sample and focused set of genesRT2 Single Primer AssaysRT2 Custom PCR Arrays

� Identify Responsible Transcription FactorsSureFind Transcriptome PCR Arrays

� MicroRNA studiesmiScript MicroRNA PCR Arrays and assays

� Assess Biological ImpactCIGNAL™ Reporters

� Analyze interaction between DNA & nuclear proteinsEpiTect Chromatin Immunoprecipitation PCR Arrays

� Quantify secreted proteins in blood plasma seraELISArrays

� Knockdown AnalysisSureSilencing shRNA Plasmids & FlexiTube and FlexiPlate siRNA

� Somatic DNA Mutation StatusqBiomarker Somatic DNA Mutation PCR Arrays

� Copy Number AnalysisqBiomarker Copy Number PCR Arrays & Assays

� New! GeneRead DNAseq Targeted Exon Enrichment Panels for N GS


www.SABiosciences.com

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www.SABiosciences.com

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Pathway central – 100+ signaling pathway maps

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QIAGEN website: Geneglobe.com: 500 pathway maps


New literature search engine - SciGlobe

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http://sabiosciences.com/sciglobe.php

SciGlobe - a more intelligent way to search the literature

SciGlobe is an advanced search engine for the biomedical literature. It uses cutting-edge linguistic technology to quickly find and deliver the most relevant search results. SciGlobe search delivers focused content, not just lists of publications.


• Complete experimental systems• RNA isolation kits, PCR Arrays, master mix, first strand kit, and free data analysis

software• Systematic controls to monitor sample quality, RT and PCR efficiency• 2 hours from RNA to data analysis

• Breadth of pathway content• Profile the genes and pathways that you really care about• More than 150 pathways for human, mouse, rat, drosophila, rhesus macaque, dog, pig• Customizable by gene, set, or pathway

• Superb real-time PCR performance• Reproducibility, Sensitivity, Specificity, Reliability

• Broad application• Cancer, immunology, neuroscience, stem cells & much more

PCR array system summary

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Thank you for attending

Thousands of scientists have discovered the power of PCR Arrays.

Join them on the road to success!

Are you ready to try? Pleasecontact us today

US and Canada Customers:Contact Technical Support: 9 AM-6 PM M – F

ESTCall: 1-888-503-3187 Email: [email protected]

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pcr array 2013

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