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Personalized Genetic Technology and Novel Multiplex Quantitative PCR Hong Kong University Tiffany Jiang Ph.D. Asia Pacific Marketing Manager Beckman Coulter At your fingertip!! Multiplex Gene Expression Sequencing STR SNP AFLP T-RFLP Methylation Other Fragment Analysis Gene Expression Cell has ~30,000 genes On average ~ 10,000 expressed in a cell 10 to 500 associated with a particular disease 10 to 50 associated with a pathway or response 10,000 1000 100 Signatures 10 # of Genes Disc overy 100 1000 10,000 100,000 # of Samples Disease Signature DrugResponse Response Drug Cancer Prognosis • Cytotoxicity and DNA damage • • Anticancer drug HepG2 cell lineresponse study • • Human ID’d a tumor/xenograft 20 gene set out model of 250 • • 1570 genes 100 differentially respond Classified compounds correctly cytotoxic anti-inflammatory and • 32asgene signature tracked potency • • • • • damaging agents ofDNA response to 7 chemotherapy agents Burczynski et al.; Tox. Sci. 58, H. Zembutsu et.al. Cancer Res 62, 399-415 (2000) 518-27 (2002) Breast cancer Human clinical breast cancer study ID’d a 70 gene set out of 25,000 Prognostic signature for metastases Outperformed other clinical parameters in predicting disease outcome van’t Veer et al., Nature 415, 530-536 (2002) Assessing Glioblastoma Prognosis Using A Multiplex RT-PCR Assay on Formalin Fixed Paraffin Embedded (FFPE) Tissue Tumor grade and survival of gliomas •The influence of tumor grade on survival is a well-recognized prognostic feature of gliomas. 38 genes appear robustly associated with survival Actual survival data Scrambled survival data 38 genes: survival in all 4 data sets (MD, UCSF, UCLA, MGH) FFPE •• Test Test needs needs to to be be amenable amenable to to routinely routinely processed, processed, clinically clinically available available tissue: tissue: –– Formalin Formalin fixed, fixed, paraffin paraffin embedded embedded specimens specimens 1000 Discovery Arrays 100 10 # of Genes 10,000 Accelerate: Multiplex PCR Signatures Multiplex PCR (eXpress Profiling) RT PCR 100 1000 10,000 100,000 # of Samples • eXpress Profiling (XP PCR) • Multiplexed low-cost process (2-40 genes) • High sensitivity (104 molecules) • 3+ log dynamic range • Low sample requirement (5 ng) • Work with small tissue samples • Work with varied quality RNA (fixed samples) • Endpoint PCR with capillary readout Key Components of GeXP • Chemistry (XP-PCR) – Patented priming strategy • PCR- based • Maintains relative gene ratios • Minimizes PCR artifacts • • • • Multiplexed low-cost process (2-40 genes) Solution-phase RT-PCR High sensitivity Low sample requirements – As little as 5 ng total RNA/reaction – Work with small clinical samples Process Overview RT Gene-specific Multiplex XP-PCR Total RNA 96-well format Sample dilution Gene Expression Analysis XP PCR Priming Process • 1: Reverse Transcription mRNA to cDNA – Reverse Chimeric Primers priming specific transcripts in Total mRNA preparation. Universal Reverse Tag sequence AAAAAAAA AAAAAAAA AAAAAAAA Gene Specific Primers XP PCR Priming Process • 2a Multiplex PCR – Step 1 of the reaction: • Universal/Gene specific primers • Cycle 1 to 3- gene specific primers work • Set gene ratios Forward Chimeric Primer with Universal Forward Primer Sequence Gene 1 cDNA Gene 2 cDNA Gene 3 cDNA Universal Reverse Primer sequence added in RT step XP PCR Priming Process • 2b Multiplex PCR – Cycles 3 through 35 – Universal primer in excess concentration take over- and amplify multiple target fragments simultaneously and uniformly. – Treats all templates as same chemical entity [ ] Universal reagents in reaction are in excess, driving amplification. Dye-Labeled Forward Universal Primer Unlabeled Universal Primer After Universal Amplification HuMulti2 SKL.A10_041223080B 80000 • Capillary Electrophoresis Separation of Fragments – by size – precise signal quantification. • No clean-up! 60000 157.70 50000 325.74 40000 172.03 30000 208.66 303.47 178.37 20000 235.75 249.93 10000 272.83 152.03 221.34 194.02 Dye Signal Unincorporated primers and fragments diluted, separated, data filtered, and ready for analysis. 214.60 70000 163.69 186.76 242.76 202.13 286.14 294.16 280.78 255.07 265.64 227.33 314.63 340.69 352.33 0 150 175 200 225 250 Size (nt) 275 300 325 350 { { { GenomeLab™ GeXP Genetic Analysis System • Size range 150-350 nt 40000 beta actin 277.96 35000 cyclophilin A 307.03 30000 341.64 • amplification products are well balanced within a linear detection range of the CE system for precise signal quantification 25000 • Reference genes are included to allow peak signal normalization for relative quantitative analysis 314.28 20000 172.82 200.44 264.43 164.85 285.90 333.68 15000 210.05 10000 150.90 GAPDH 245.69 181.13 224.25 272.05 250.07 • Gene-specific peaks -identified, quantitated and normalized 298.97 290.92 324.73 228.51 5000 158.46 238.99 195.68 171.65 257.25 188.55 276.87 305.89 349.81 0 150 175 200 225 250 Size (nt) 275 300 325 350 • Gene expression profiles - created and visualized using proprietary software. FFPE Assay Design • short amplicons: <75 bp • variable length in chimeric primers • performed and optimized by Althea, Inc. • two multiplexes - total 24 genes AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA Chimeric Chimeric primers primers HuMulti2 SKL.A10_041223080B 80000 Kan(r) Kan(r) 60000 214.60 Separation between between peak peak {{ Separation Minimum Size Size {{ Minimum 70000 157.70 50000 40000 172.03 208.66 178.37 20000 Maximum Size Size {{ Maximum 30000 325.74 303.47 235.75 249.93 10000 272.83 286.14 152.03 294.16 221.34 280.78 194.02 255.07 242.76 163.69 Dye Signal 265.64 227.33 202.13 186.76 352.33 314.63 340.69 0 150 175 200 225 250 Size (nt) 275 300 325 350 Developing ’38 genes’ into a useful test • develop score: summarize expression profile into a single number (like PSA score) • simplify: reduce genes from 38 • adapt: for clinical use needs to work with clinical samples • accelerate: to achieve sufficient throughput for clinical use • validate: test in independent sample set • clinical samples being treated with current standard of care Case VII: Single Cell Analysis Automation Flow GeXP AmpliGrid ̶ Single cell gene expression profiling Detecting multiple genes from a single cell without pre-amplification HuRef Multiplex on Breast cancer cell No pre-amplification, no bias in the final results Fluidigm system pre-amplify targets with 35 cycles Induced Pluripotent Stem cell Skin cells Isolation Patients Genetic Diseases iPS Reprogramming Expansion Personalized Stem Cell Therapies Differentiation Autologous Transplantation hC C N hG G1 AP D H hP H B hT BP hc M yc hK lf4 hO ct hS 4 O X hN 2 an og hL hD in2 N 8 M T hD 3B PP hD A2 PP A hG 4 D F hG 3 U S hM B YB L hP 2 H C hS 1 A hS LL4 TE L hT LA ER Tv hZ 1 M FP4 Xs 2 M hOc Xs t4 -h So M X s x2 h M Xs Klf4 M -hc Xs M -h y c M Nan Xs o -h g LI N 28 Normalized Values Automation 12 10 CA-1 hESC BJ-pMXs 4YA hiPSC GeXP 8 6 4 2 0 Flow Vehicle Control 3 Rosiglitazone 100 uM Troglitazone 100 uM 2 * 1 Genes B Ppi et a aac tin 4 H o1 C yp 1A 1 C yp 1A 2 C yp 2B C yp 1 (M C yp 1) (P B C 1) yp 3A 1 C yp 4A C yp 1 2D 22 N A D Cyp PH 2 C E1 yp re d U dp . gt r2 A ld h N qo 1 p5 3 G ad d4 G ad 5 d1 53 C ox -2 C as p3 Pc n C yc a lin D 1 p2 1 Gene Expression Ratio (relative to Gapdh) Gene Expression Profile Obtained in Glitazone -Treated Rat Primary Hepatocytes * Pioglitazone 100 uM * * * 0 Apoptosis pathwaysmall cell lung cancer Biotech J.com 2002 40-gene Multiplex Panel on GeXP Additional References Additional References Additional References Inspiration Dedicated to improving patient health and reducing the cost of care.