Pcr array 2013
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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]
Sample & Assay Technologies
Pathway-focused solution for expression analysis- Complete experimental systems: sample prep through data analysis
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SABiosciences.com
Sample & Assay Technologies
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
Sample & Assay Technologies
Pathway-focused expression analysis- Complete Experimental Systems: Sample Prep through Data Analysis
Genes (mRNA)
microRNA (miRNA)
DNA Methylation
Cancer Mutation
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Sample & Assay Technologies
Gene expression regulates biology
All require molecular signaling for action
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Sample & Assay Technologies
Biological markers define processes
Cell cycle Angiogenesis
p53MDM2CyclinsCDKs
VEGFbFGF
ANGPTPDGF
IL8TLRIFNγTNFβIn
divi
dual
pa
rtic
ipan
ts
Inflammation
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Sample & Assay Technologies
Pat
hway
Cell cycle Angiogenesis Inflammation
Complete biological story- Built on pathway / network analysis
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Sample & Assay Technologies
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:
Sample & Assay Technologies
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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Sample & Assay Technologies
All pathways @ www.SABiosciences.com
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Sample & Assay Technologies
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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Sample & Assay Technologies
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
Sample & Assay Technologies
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
Sample & Assay Technologies
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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Sample & Assay Technologies
How genes on PCR arrays are selected- Published sources
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http://gncpro.sabiosciences.com/gncpro/gncpro.php
Sample & Assay Technologies
Relevant pathway content on PCR arrays
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Sample & Assay Technologies
Contents 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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Sample & Assay Technologies
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
Sample & Assay Technologies
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
Sample & Assay Technologies
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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Sample & Assay Technologies
• 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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Sample & Assay Technologies
• 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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Sample & Assay Technologies
• 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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Sample & Assay Technologies
Topics will be covered
1. Introduction to the PCR Arrays
2. How PCR Arrays Work
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
4. Applications for PCR Arrays
5. miRNA PCR Arrays & qBiomarker DNA Mutation PCR Arra ys & EpiTect DNA Methylation PCR Arrays
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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
1. Introduction to the PCR Arrays
2. How PCR Arrays Work
3. Performance of PCR Arrays
4. Applications for PCR Arrays
5. What’s next after PCR Arrays
Topics will be covered
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Sample & Assay Technologies
• 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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Sample & Assay Technologies
� 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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Sample & Assay Technologies
• 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
24 Genes : 4 Samples
96-well 384-well
32 Genes : 3 Samples
48 Genes : 2 Samples
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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Sample & Assay Technologies
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
Sample & Assay Technologies
www.SABiosciences.com
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Sample & Assay Technologies
www.SABiosciences.com
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Sample & Assay Technologies
Pathway central – 100+ signaling pathway maps
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QIAGEN website: Geneglobe.com: 500 pathway maps
Sample & Assay Technologies
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.
Sample & Assay Technologies
• 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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Sample & Assay Technologies
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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