Personalized Genetic Technology and Novel Multiplex Quantitative PCR

Transcription

1 Personalized Genetic Technology and Novel Multiplex Quantitative PCR Hong Kong University Tiffany Jiang Ph.D. Asia Pacific Marketing Manager Beckman Coulter

2 At your fingertip!! Multiplex Gene Expression Sequencing STR SNP AFLP T-RFLP Methylation Other Fragment Analysis

3 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

4 Disc overy Signatures , ,000 # of Samples # of Genes ,000

5 Disease Signature Drug Drug Response Response Cytotoxicity and DNA damage Anticancer HepG2 cell drug line response study Human ID d a tumor/xenograft 20 gene set out model of Classified genes 100 differentially compounds respond correctly 32 as gene cytotoxic signature anti-inflammatory tracked potency and of DNA response damaging to 7 chemotherapy agents agents Burczynski et al.; Tox. Sci. 58, H. Zembutsu et.al. Cancer Res 62, (2000) (2002) Cancer Prognosis 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, (2002)

6 Assessing Glioblastoma Prognosis Using A Multiplex RT-PCR Assay on Formalin Fixed Paraffin Embedded (FFPE) Tissue

7 The influence of tumor grade on survival is a well-recognized prognostic feature of gliomas. Tumor grade and survival of gliomas

8 38 genes appear robustly associated with survival Actual survival data Scrambled survival data

9 38 genes: survival in all 4 data sets (MD, UCSF, UCLA, MGH)

10 FFPE Test needs to be amenable to routinely processed, clinically available tissue: Formalin fixed, paraffin embedded specimens

11 # of Genes ,000 Arrays Accelerate: Multiplex PCR RT PCR Discovery Multiplex PCR (express Profiling) , ,000 #of Samples Signatures 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

12 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

13 Process Overview RT Gene-specific Multiplex XP-PCR Total RNA 96-well format Sample dilution Gene Expression Analysis

14 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 Gene Specific Primers AAAAAAAA

15 2a Multiplex PCR Step 1 of the reaction: Universal/Gene specific primers Cycle 1 to 3- gene specific primers work Gene 1 cdna Set gene ratios XP PCR Priming Process Forward Chimeric Primer with Universal Forward Primer Sequence Gene 2 cdna Gene 3 cdna Universal Reverse Primer sequence added in RT step

16 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

17 After Universal Amplification HuMulti2 SKL.A10_ B Capillary Electrophoresis Separation of Fragments by size precise signal quantification. No clean-up! Unincorporated primers and fragments diluted, separated, data filtered, and ready for analysis. Dye Signal Size (nt) { { {

18 GenomeLab GeXP Genetic Analysis System Size range nt beta actin cyclophilin A amplification products are well balanced within a linear detection range of the CE system for precise signal quantification Reference genes are included to allow peak signal normalization for relative quantitative analysis GAPDH Gene-specific peaks -identified, quantitated and normalized Size (nt) Gene expression profiles - created and visualized using proprietary software.

19 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 Chimeric primers

20 HuMulti2 SKL.A10_ B Minimum Size { Separation between peak { Kan(r) Maximum Size { Dye Signal Size (nt)

21 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

22 Case VII: Single Cell Analysis Automation Flow AmpliGrid GeXP

23 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

24 Skin cells Induced Pluripotent Stem cell Isolation Reprogramming ips Patients Genetic Diseases Personalized Stem Cell Therapies Expansion Autologous Transplantation Differentiation

25 htertv1 hzfp42 MXs-hOct4 MXs-hSox2 MXs-hKlf4 MXs-hcMyc MXs-hNanog MXs-hLIN28 hstella CA-1 hesc BJ-pMXs GeXP 4YA hipsc Automation Flow hoct4 hsox2 hnanog hlin28 hdnmt3b hdppa2 hdppa4 hgdf3 hgusb hmybl2 hphc1 hsall4 hklf4 hcmyc htbp hphb hgapdh hccng1 Normalized Values

26 Cyp1A2 Cyp2B1 Cyp (M1) Cyp (PB1) Cyp3A1 Cyp4A1 Cyp2D22 Cyp2E1 NADPH Cyp red. Udpgtr2 Aldh Nqo1 p53 Gadd45 Gadd153 Cox-2 Casp3 Pcna CyclinD1 p21 Ho1 Ppia Beta-actin Gene Expression Profile Obtained in Glitazone -Treated Rat Primary Hepatocytes Vehicle Control Pioglitazone 100 um Rosiglitazone 100 um Troglitazone 100 um * * * * * Cyp1A1 Genes Gene Expression Ratio (relative to Gapdh)

27 Apoptosis pathwaysmall cell lung cancer Biotech J.com 2002

28 40-gene Multiplex Panel on GeXP

29 Additional References

30 Additional References

31 Additional References

32 Inspiration Dedicated to improving patient health and reducing the cost of care.