Real-Time PCR Applications and Genomic Assays. Absolute Quantification. Is there any (amplifiable) DNA? How much DNA is there?

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1 Real-Time PCR Applications and Genomic Assays Absolute Quantification Is there any (amplifiable) DNA? How much DNA is there? 2

2 Microorganism Quantitation Type Viruses Bacteria Fungi Parasites Examples Plant, animal, human. Any bacteria including dead, slow growers, spores... Yeasts and molds. Toxoplasma and others. Absolute quantification In research 3 Gene Expression Studies IL-1 ALPHA IL-1 BETA IL-2 IL-4 Calibrator IL-8 IL-5 Cyto k in e Ge n e Ex p re s s io n Ta rge t Target Genes IL-10 IL-12p35 IL-12p40 IL-15 F D c a lib ra to r Samples IFNg H S a m p le 4 TNF-ALPHA

Gene Expression Studies Is my gene of interest differently expressed - in different tissues? - between healthy and tumor cells? - over time or treatment? How reliable are the results of my array?

3 Gene Expression Studies Is my gene of interest differently expressed - in different tissues? - between healthy and tumor cells? - over time or treatment? How reliable are the results of my array? 5 Tools to perform Gene Expression Studies - Databases: GeneAssistTM Pathway Atlas What is GeneAssistTM Pathway Atlas? 7 GeneAssist TM Pathway Atlas provides a set of more than 350 easy to understand signal transduction, metabolic and disease state pathway maps and descriptions in an interactive interface which links AB s gene-specific tools for studying each pathway component.

Tools to perform Gene Expression Studies - Databases: PANTHER Classification System http://www.pantherdb.

display technology 8 Tools to perform Gene Expression Studies - TaqMan Gene Expression Assays It s 2008, why are you still designing your own primers and probes?

4 Tools to perform Gene Expression Studies - Databases: PANTHER Classification System Login to the PANTHER Database System Have full access to previously Celera-Only data Human, Mouse & Rat Genomes Drosophila melanogaster Get access to Enhanced MapViewer Advanced real-time display technology 8 Tools to perform Gene Expression Studies - TaqMan Gene Expression Assays It s 2008, why are you still designing your own primers and probes? Human Mouse Rat Canine Macaques Zebrafish Arabidopsis Drosophila C.elegans Mitochondria Ready-to-use, off-the-shelf Assays Single tube format Run under universal cycling conditions For Gene Expression Studies: More than 700,000 genespecific TaqMan probe and primer sets Pre-Designed Pre-Optimized 9

µl reactions TaqMan MGB Probe(s) and two primers, 20x mix Includes CD with an assay

5 TaqMan Assays Massive Bioinformatics Pipeline - High Content 10 TaqMan Gene Expression Design based on NCBI RefSeq Database and CELERA DISCOVERY SYSTEM Single tube formulation: 250 x 20 µl reactions TaqMan MGB Probe(s) and two primers, 20x mix Includes CD with an assay information file Works with universal cycle conditions 11 No assay design, formulation or optimization!

assay whose primers and probes are designed within a single exon. Such assays will, by definition detect genomic DNA. indicates an assay that may detect genomic DNA.

6 Important information for different gene expression applications Assay specificity: genomic DNA, mrna, homology with other genes,... The gene expression Assay ID suffix indicates the assay specificity: _m1 _s1 _g1 _mh, _sh, _gh _u indicates an assay whose probe spans an exon junction and will not detect genomic DNA indicates an assay whose primers and probes are designed within a single exon. Such assays will, by definition detect genomic DNA. indicates an assay that may detect genomic DNA. The assay primers and probe may also be within a single exon Assay ID indicates that the assay was designed to a transcript belonging to a genefamily with high sequence homology. The assays have been designed to give between 1,000 30,000-fold greater discrimination from the closest homologous transcript indicates an assay whose amplicon spans an exon junction and the probe sits completely in one of the spanned exons 12 Important information for different gene expression applications Amplicon size: fragmented RNA, for example from FFPE. 13 Amplicon Size

Important information for different gene expression applications Silencing: Ambion sirnas silence the expression of the NCBI RefSeq mrnas interrogated by the listed TaqMan Gene Expression assays

7 Important information for different gene expression applications Silencing: Ambion sirnas silence the expression of the NCBI RefSeq mrnas interrogated by the listed TaqMan Gene Expression assays Ambion sirnas 14 Important information for different gene expression applications Public Exon-Exon Spanning & Assay Location Assay coverage: transcripts covered by this assay design (different isoforms). RefSeq and GenBank links Celera Exon-Exon Spanning & Assay Location Celera Information 15

Ready-to-use plate with your assays of interest! High throughput: analysis of high number of samples and/or genes.

8 Important information for different gene expression applications Gene details: functional and biological information PANTHER Classification GO Categories 16 Important information for different gene expression applications TaqMan Low Density Array Ready-to-use plate with your assays of interest! High throughput: analysis of high number of samples and/or genes. 384 spots, 2 µl volume 1 to 8 Samples 18 Pre-Made TaqMan Gene Expression Assays pre-loaded 12 to 381 Genes (per one array) Number of Replicates: 1, 2, 3 & 4 per card

9 What is a translocation and why is that important? Translocations can cause Cancer Example: chronic myeloid leukemia (CML) & Philadelphia Chromosome Sometimes a fusion transcript between two genes occurs (e.g. BCR-ABL) Fusion transcripts are complex, and difficult to study 19 Entrez Probe Shows All TaqMan Assays Mapped to Gene & Transcripts Search Entrez Probe Identify gene/assay of interest Click through to AB Web site and order TaqMan Assays recognize different splice variants 23

10 Copy Number Variations and Gene Dosage effects From Nature, Vol. 437 (Oct. 20, 2005) CNV is one of the important structural variation in the genome CNV may account for a significant proportion of normal variation and also disease association Genetic diseases, cancer, neurological disorders, and immune diseases CNVs has impacts on levels of gene expression and therefore produces gene dosage effects Gene dosage effect could be phenotypic DME gene CYP2D6 in drug metabolizing CCL3L1 effects the susceptibility to HIV/AIDS 25 TaqMan Gene Dosage Assays 5 TaqMan Gene Expession assays available HsXXXXXXXX_cn They are a valuable complement to TaqMan SNP GT assays Accurate, robust, reproducible and specific Assay ID Mapped ID Availability Gene Symbol Hs _cn GSTM1 Inventoried GSTM1 Hs _cn GSTT1 Inventoried GSTT1 Hs _cn CYP2E1 Inventoried CYP2E1 Hs _cn CYP2A6 Inventoried CYP2A6 Hs _cn CYP2D6 Inventoried CYP2D6,hCG

11 Quantifying MicroRNA Why study microrna? What if expressed mrna species measured by real-time PCR are rendered non-functional by binding cognate microrna? Thus no protein produced from expressed mrna... Application/publication areas of microrna studies: Cell proliferation Metabolism Cell death Stem cell & neuron development Cancer DNA methylation and chromatin modification 27 Quick Terminology Review RNAi (RNA interference) Mechanism naturally occurring in cells, where double stranded RNA is degraded mirna (microrna) ~22 nt single-stranded RNAs processed from longer hairpin precursors. Occur naturally in cells (endogenous) and affect gene regulation through the RNAi pathway. mirnas bind to mrna and silence genes mainly through translational repression, but also through mrna degradation. sirna (short interfering RNA) ~21-23 nt double-stranded RNAs that guide the cleavage and degradation of their cognate RNAs. Arise in cell either through cleavage of long double stranded RNA by the enzyme Dicer or by introduction through cell transfection (manual intervention). 29

12 MicroRNAs: Genomics Short, single-stranded, endogenous, non-coding RNAs Cleaved from nt pri-mirna precursors by Drosha/Dicer Nucleus Cytoplasm 5 Drosha Dicer C C U C 3 31 pri-mirna pre-mirna Mature mirna TaqMan MicroRNA Assays Design based on Sanger Registry: mirbase Includes separate assay-specific 5x reverse transcriptase primer and 20x TaqMan MGB FAM -probe and primers 150 x 20 µl reactions as single assays or compiled as panels Human assays and 10 endogenous controls Assays for mice and rats Recent assays made for Arabidopsis, C. elegans, and Drosophila Use with TaqMan MicroRNA RT Kit and Universal Master Mix Includes CD with an assay information file Works with universal cycle conditions No assay design, formulation or optimization! 32

13 Applications Real-Time Applications Absolute Quantification Relative Quantification End-Point Applications Plus/minus Allelic Discrimination 35 Allelic Discrimination What is an SNP? Allele 1 5 -GTCGTACGTCAGTCCG-3 3 -CAGCATGCAGTCAGGC-5 Allele 2 5 -GTCGTACTTCAGTCCG-3 3 -CAGCATGAAGTCAGGC-5 Single Nucleotide Polymorphism (SNP) Allelic frequency of minor allele > 1% Usually two alleles present ( bi-allelic markers ) 36

14 Why to work with SNPs (bi-allelic markers) instead of more polymorphic markers? Very high density of markers Lower mutation rates for SNPs than for microsatellites Simple analysis (allele calling) Easier to automate Yes/No for each of two alleles Can be functional 37 Why Investigate SNPs? SNPs used as genetic markers Linkage disequilibrium Recombination Association studies LD mapping of complex traits Fine mapping Demographic events Identifying candidate genes Study selection SNPs with biological effect Drug metabolism ( Pharmacokinetics ) e.g. Cytochrome p450 Direct cause of disease e.g. Factor V Leiden, sickle-cell anemia 38

15 Basic technologies for SNP Genotyping Assay Mechanism for Allelic Discrimination Simple PCR (allele-specific PCR) Real Time PCR Allele-specific PCR (SYBR Green) TaqMan probes 43 Applications Real-Time Applications Absolute Quantification Relative Quantification End-Point Applications Plus/minus Allelic Discrimination 47

16 Plus / Minus Calling in Conventional PCR or? 48 Presence or Absence of a Plasmid / Transgene Toxin gene, antibiotic resistance TRANSGEN Is the transgene in the mouse? Colony screening

17 Critical points to obtain a successful result: Time to result: faster than conventional methods Standardized protocol: facilitate workflows and generate consistent results for different detections Specificity: capacity to differentiate as many species as possible Sensitivity: establish the lowest limit of detection with maximum efficiency Accuracy: fewer false negatives and false positives Higher repeatability and reproducibility 50 Include Internal Positive Control (IPC) The IPC consists of a template, a primer set, and a dye labeled probe added to each well of a reaction plate. Included for two reasons: to detect PCR inhibition (reduces number of false negatives) to demonstrate that kit amplifies IPC-signal designed to be independant from target startingcopy number IPC signal won t be influenced significantly if Ct of target is >20 51

18 Applied Markets Division in Applied Biosystems Markets Forensic Biosecurity / Public Health Quality & Safety Testing Food Beverage Agriculture Pharma Quality Environ Control mental Applications DNA-based Human Identification Toxicology/Drug Screening Surveillance and Detection of Threats: Bioterrorism and Outbreaks of Emerging Infectious Diseases Chemical threats Food Quality Safety: Pathogens & Contaminant testing Identify Sterility Isolates Biologics Water Quality and Safety: Pesticides, Perchlorate Increasing Validation & Regulation 52 Solutions for Regulated Environments Integrated solutions provide Higher Safety and Reliability Easier Handling Faster Adoption Product range is constantly increasing...for more information please ask us! 53

19 RNA Interference What is RNAi? RNAi means RNA interference A phenomenon in which the introduction of double-stranded RNA (dsrna) into a diverse range of organisms and cell types causes degradation of the complementary mrna. A tool to: Analyze gene function Study pathways Identify and validate drug targets 61

20 Mechanism of RNAi short-interfering ~21 bp dsrna RNA Induced Silencing Complex 62 sirna Experimental Workflow sirna Design sirna Synthesis sirna Delivery sirna Assay Successful sirna experiment 63

21 Steps of an RNAi Experiment STEP STEP STEP STEP STEP 1 Obtain effective sirnas Optimize sirna delivery Test sirna silencing efficiency Examine biological impact of silencing the target Correlate silencing with biology 64 Legal Statements For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: PLEASE REFER TO THE USER'S GUIDE OR PACKAGE INSERT OF THE PRODUCTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Applera, Applied Biosystems, AB (Design), ABI PRISM, Primer Express, and VIC are registered trademarks and FAM and CELERA DISCOVERY SYSTEM are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. Ambion,GeneAssist, and Silencer are registered trademarks of Ambion, Inc.in the U.S. and/or certain other countries. All other trademarks are the sole property of their respective owners. For Research Use Only. Not for use in diagnostic procedures. Pathogen detection products are not sold in the United States for surveillance or other public health epidemeology purposes. MicroSeq Bacterial and Fungal Identification Kits: NOTICE TO PURCHASER: LIMITED LICENSE Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased solely in the environmental testing, quality control/quality assurance testing, food and agricultural testing applications, and also for the purchaser s own internal research. No other patent rights (such as 5 Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TaqMan Pathogen Detection Kits: Please refer to the respective TaqMan Pathogen Detection Kits User s guide or product insert for limited label license or disclaimer information. Applera, Applied Biosystems, AB (Design), and MicroSeq are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. Ambion is a registered trademarks and MagMAX is a trademark of Ambion, Inc., an Applied Biosystems Business. TaqMan is a registered trademark of Roche Molecular Systems, Inc Applied Biosystems. All rights reserved.

Real-Time PCR Validations

Real-Time PCR Validations Relative gene expression Example experiment: I have 2 samples: untreated and treated. Question: What happens to the expression of gene X when I treat the cells? Answer: Expressed