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Overview on Agilent Lab on a Chip Applications

Overview on Agilent Lab on a Chip Applications. 2100 Bioanalyzer Solutions March 2007. The Lab-on-a-Chip Approach. Active Control of Fluids without Moving Parts. Sample volumes 1 -4 µl 10 -12 samples depending on Assay Separation, staining, detection of samples

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Overview on Agilent Lab on a Chip Applications

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  1. Overview on Agilent Lab on a ChipApplications 2100 Bioanalyzer Solutions March 2007 Group/Presentation Title Agilent Restricted

  2. The Lab-on-a-Chip Approach Active Control of Fluids without Moving Parts Sample volumes 1 -4 µl 10 -12 samples depending on Assay Separation, staining, detection of samples Results in 5-30 minutes available No extra waste removal needed Disposable Chip, no crosscontamination Group/Presentation Title Agilent Restricted

  3. Lab-on-a-Chip or Gel - A Faster Answer ! Group/Presentation Title Agilent Restricted

  4. Agilent 2100 Kits and consumables Various Kits and consumables are available to support the predefined methods o the 2100 Bioanalyzer Group/Presentation Title Agilent Restricted

  5. RNA Applications Gene Expression RNA QA/QC for Microarrays Genomic DNA Contamination RNA QA/QC for qPCR smallRNA QA/QC RNA QA/QC for mPCR Group/Presentation Title Agilent Restricted

  6. Specifications RNA assays (Series2 assays) RNA 6000 Nano LabChip kit RNA 6000 Pico LabChip kit Group/Presentation Title Agilent Restricted

  7. total RNA determine integrity and quality of total RNA determination of RNA concentration identify ribosomal peaks calculate the ratio of ribosomal peaks (18S/28S or 16S/23S) RNAintegrity number(RIN) mRNA determine integrity and quality of mRNA samples Determination of mRNA concentration calculate % ribosomal RNA in mRNA samples Features of the RNA 6000 Assays Group/Presentation Title Agilent Restricted

  8. 11 18S 28S 10 9 8 7 marker 6 Fluorescence 5 4 3 2 1 0 18S 28S 19 24 29 34 39 44 49 54 59 64 69 Time (seconds) 11 10 9 8 7 6 marker 5 Fluorescence 4 3 2 1 18S 28S 0 19 24 29 34 39 44 49 54 59 64 69 Time (seconds) RNA 6000 Nano LabChip kit Analysis of Total RNA Integrity Typical first QC step after RNA sample prep prior to microarrays or real-time PCR High quality total RNA Partially degraded total RNA 2100 bioanalyzer: single lane gel-like image 2100 bioanalyzer: electropherogram Group/Presentation Title Agilent Restricted

  9. RNA QC in Routine Gene Expression Workflow Cells / Culture RNA isolation Start again with sample isolation Total RNA RNA QC via Agilent 2100 bioanalyzer RIN RIN above threshold Continue with downstream Experiment (Microarray, real-time PCR, etc.) Group/Presentation Title Agilent Restricted

  10. 12.5 10.0 7.5 Fluorescence 5.0 2.5 0.0 18S 28S 20 15 Fluorescence 10 5 0 18S 28S 80 70 60 50 Fluorescence 40 30 20 10 18S 28S 0 19 24 29 34 39 44 49 54 59 64 69 Time (seconds) RIN Application – Directly Compare Samplessame sample in different dilutions • 25 ng/µl: RIN 8 When testing an identical RNA sample in various dilutions, identical RINs are obtained – within narrow limits • 100 ng/µl: RIN 8 108 samples 3 dilutions CV RIN: 3 % CV ribosomal ratio: 22 % • 500 ng/µl: RIN 8 Group/Presentation Title Agilent Restricted

  11. 45 40 35 30 Fluorescence 25 20 9 8 15 7 10 6 Fluorescence 5 5 4 3 18S 28S 0 2 1 0 18S 28S 3.5 3.0 2.5 2.0 Fluorescence 1.5 1.0 0.5 0.0 19 24 29 34 39 44 49 54 59 64 69 Time (seconds) RIN Application – Assessment of RNA Integrity • Intact RNA: RIN 10 • Partially degraded RNA: RIN 5 • Strongly Degraded RNA: RIN 3 Group/Presentation Title Agilent Restricted

  12. Mouse kidney II mRNA Mouse kidney I mRNA Bovine kidney mRNA Mouse liver mRNA Mouse testis mRNA RNA 6000 ladder Rat brain mRNA Ribosomal RNA contamination in mRNA samples rRNA contamination: 19.5 % rRNA contamination: 1.7 % Group/Presentation Title Agilent Restricted

  13. 200 pg total RNA 18S 28S 24 29 34 39 44 49 54 59 64 69 Laser Microdissection – PALM MicroBeam System A Laser microdissection B Laser pressure catapulting: Section after catapulting of selected area C Catapulted area in the collection device RNA extraction RNA sample QC using the Agilent 2100 bioanalyzer and the RNA 6000 Pico LabChip kit Laser Microdissection and Pressure Catapulting (LMPC) Group/Presentation Title Agilent Restricted Data kindly provided by P.A.L.M. Microlaser Technologies

  14. Cy3/ Cy5 Labeling mRNA QC Total RNA QC cRNA fragmentation Data evaluation cRNA Hybridization - Workflow Array experiment Group/Presentation Title Agilent Restricted

  15. Series II RNA Assays • A new RNA Ladder is now included in the kit. • Improved quantitation accuracy with salt containing samples. No late migration of 28S peaks • Pico: higher sensitivity, better reproducibility and accuracy in quantitation • Higher reagent volumes at no charge old new Group/Presentation Title Agilent Restricted

  16. RNA Con’t • Increased sensitivity, Pico Assay • 50 pg/µL total RNA in water (Series II RNA 6000 Pico assay) Group/Presentation Title Agilent Restricted

  17. Analysis of Small RNA (using RNA 6000 Assay) • Small RNA fraction: < 200 nts e.g. miRNA, siRNA, snRNA, tRNA, 5S RNA • Bioanalyzer allows discrimination of different profiles Group/Presentation Title Agilent Restricted

  18. Small RNA Assay specifications • Analytical Range                  6 -150 nt (to avoid overlap) • Sensitivity 50 pg/µl   • (diluted Ladder - 40 nt fragment; S/N > 3:1) • Quantitative range 50 pg/µl – 2000 pg/µl • (purified miRNA in water after extraction ~<200nt) • Quantitation Reproducibility 25 % CV • (defined on Ladder) • Max amount total RNA 100 ng/µl total RNA • Carryover         Below detection limit Group/Presentation Title Agilent Restricted

  19. Lower Marker Small rRNAs [nt] miRNA tRNA New Small RNA Assay Two complimentary assays, one to evaluate total RNA integrity and the second to characterize the small RNA fraction 28s rRNA 18s rRNA Lower Marker50bp tRNA RNA 6000 Assay Small RNA Assay Fragments larger than 200nt (i.e. 18S/28S) are not injected and do not interfere with the assay Group/Presentation Title Agilent Restricted

  20. New Small RNA Assay versus existing RNA Assay 18s 28s Small RNA Region Lower Marker RIN: 8.1 RNA 6000Nano Size range: 25-6000nt Results: Integrity, Total RNA amount, gDNA contamination RNA 6000Nano kit Small RNA Kit Small RNA Region 5.8s miRNA Region 5s Lower Marker NEW! Small RNASize range: 6-150nt Results: miRNA amount, Ratio and amount of other Small RNA tRNA Group/Presentation Title Agilent Restricted

  21. Protein Applications Protein QA/QC Antibody QA/QC Compliant Protein & Antibody QA/QC Protein Expression Food Analysis Protein Purification Group/Presentation Title Agilent Restricted

  22. Protein LabChip Kits – Specifications (Series II) • • Automated analysis of 10 protein samples in less than 30 minutes • • Two assays for complementary size ranges • – Protein 80: 5 to 80 kDa – Protein 230: 14 to 230 kDa • • Sizing resolution of 10% across the size range • • Large linear dynamic range (e.g. from 15 - 2000 ng/l CA-II in PBS) • • Sensitivity equivalent to non-colloidal Coomassie (R-250) stain • • Relative and absolute quantitation • • Compatible to a variety of sample buffer components Group/Presentation Title Agilent Restricted

  23. Protein Series II Assay - Specs Group/Presentation Title Agilent Restricted

  24. detection X If no dilution was done the micelles would result in high background and low sensitivity SDS + dye destain detection protein micelles low background good signal to noise ratio SDS conc. below CMC Staining, Destaining and Detection Staining, Destaining and Detection Group/Presentation Title Agilent Restricted

  25. SDS + dye protein micelles destain detection low background good signal to noise ratio SDS conc. below CMC Staining, Destaining and Detection Staining, Destaining and Detection Group/Presentation Title Agilent Restricted

  26. Protein LabChip Applications Cell lysates • identification of over-expressed proteins • comparison of different expression patterns Column fractions • monitoring of protein isolation and purification process • check fractions for impurities Purified proteins • monitoring of impurities in protein preps • integrity check for monoclonal or polyclonal antibodies (antibody QC) Group/Presentation Title Agilent Restricted

  27. Clone Selection based on Protein Expression Example measured with Protein 50 kit colony 1 colony 2 kDa 60 53.0 50 40 32.5 Fluorescence 29.0 protein of interest 30 21.5 20 14.4 6.0 10 0 1* 8* 2 3 4 5 6 7 20 25 30 35 40 45 50 55 Time (seconds) Group/Presentation Title Agilent Restricted

  28. GFP Fusion Protein Analysis cell lysate flow through (fraction 66) wash (fraction 69) elution (fraction 77) wash (fraction 78) Courtesy of P. Sebastian and S.R. Schmidt GPC-Biotech AG, Martinsried, Germany 15 20 25 30 35 40 45 Time (seconds) Monitoring of Protein Purification Process Example measured with Protein 200 plus kit 2100 bioanalyzer: electropherogram 2100 bioanalyzer: gel-like image Group/Presentation Title Agilent Restricted

  29. Protein P230 Series II Assay • Assay reworked for optimized stability • Sizing range from 14-230 kDa • UM at 240 kDa • New lower marker and optimized LM detection (less overlap with system peak) • New ladder • Baseline stability and better sensitivity High purity UM and ladder improves usability in QC (optimized for low level impurity quantification -> compliance). Group/Presentation Title Agilent Restricted

  30. Quality Control of Antibodies Antibody analysis under reducing and non-reducing conditions Ab non-reduced Ab reduced heavy chain Ab reduced intact antibody light chain Determine the half antibody content in IgG preparations intact antibody Ab non-reduced light + heavy chain 16% half antibody 90 kDa 160 kDa Absolute Quantitation of IgG samples Group/Presentation Title Agilent Restricted

  31. Protein 80 Series II assay • sizing range 5-80 kDa (specific fit to antibody QC) • new ladder & new UM • improvements in sensitivity (sizing/quantitation) • improvements in sensitivity specifications as P230 ladder run PBS blank run Group/Presentation Title Agilent Restricted

  32. 2100 Bioanalyzer Compliance • 2100 expert software • - One version for all assays • Declaration of system validation • 2100 expert security pack • 21 CFR part 11compliance • Electronic records • - Electronic signatures • - Audit trails • 2100 bioanalyzer • - IQ and OQ/PV services • Declaration of conformity Chips and reagents - Declaration of conformity Group/Presentation Title Agilent Restricted

  33. DNA Applications Forensic Testing Food Analysis mtDNA Screening Restriction Digest Analysis qPCR validation, impurity check Gene Expression mPCR validation, impurity check Oncology Clinical Research Group/Presentation Title Agilent Restricted

  34. Application Areas for the DNA Assays • PCR product purity • Multiplex PCR Applications • Gene expression analysis via RT-PCR (target validation) • GMO testing • Pathogen detection (homeland defense, hospitals, environmental) • Genotyping applications • Duplications/ deletions • Allele frequency • Bacterial sub-typing • Forensics • Cancer diagnostics Group/Presentation Title Agilent Restricted

  35. 900, 1000 bp 473, 500 bp marker 900, 1000 bp 300, 315 bp 473, 500 bp 100, 105 bp 300, 315 bp 100, 105 bp 25 bp 25 bp marker 2 % agarose gel stained with Ethidiumbromide Data Format - Gel-Like Image c/w Agarose Gel 2100 bioanalyzer data Gel-like image Group/Presentation Title Agilent Restricted

  36. 1 2 3 4 5 Determination of PCR Product Impurity Agilent 2100 bioanalyzer Agarose gel 1 2 3 4 5 3000 bp 2000 1200 2000 800 1200 800 400 400 200 100 300 bp 200 100 Group/Presentation Title Agilent Restricted

  37. Determination of PCR Product Impurity 300 bp Quantitative data from Agilent 2100 bioanalyzer Samplec (DNA) main peak 300 bp PCR 41.4 ng/ul 40.7 ng/ul 300 bp PCR 1:4 9.6 ng/ul 9.6 ng/ul Impurity level : < 2% Quantitative data from Agilent 2100 bioanalyzer Samplec (DNA) main peak 3000 bp PCR 61.9 ng/ul 40.7 ng/ul 3000 bp PCR 1:4 14.8 ng/ul 9.8 ng/ul 3000 bp Impurity level : > 50% Group/Presentation Title Agilent Restricted

  38. GMO Detection: Determination of GM Soya Percentage EPSPS gene target: specific for Roundup Ready GM soya (Monsanto) Soya lectin gene target Data kindly provided by CCFRA Group/Presentation Title Agilent Restricted

  39. Effect of Multiplex PCR buffer on a 19-plex PCR Data kindly provided by QIAGEN GmbH, Germany Group/Presentation Title Agilent Restricted

  40. Tumor Diagnostics • Spiking experiment with given amount of cancer cells • Enrichment with AdneGen Cancer Select kit (antibody based immunomagnetic enrichment.) • Multiplex Amplification with AdnaGen CancerDetect kit • Detection with Agilent 2100 Bioanalyzer and DNA 500 LabChip kit Data kindly provided by Adnagen Group/Presentation Title Agilent Restricted

  41. Detection of Single Base Mutations (1) in Exons 7 and 8 of the Human p53 Gene by RFLP Mapping using the DNA 7500 kit exon 7 exon 8 Amplify exons 7 and 8 (resulting products: 618 bp fragment and 200 bp fragment) Digest with Hpa II In each example one of the restriction sites can be deleted by a point mutation 83 bp, 91 bp, 168 bp, 276 bp 91 bp, 109 bp Analyze using Agilent 2100 bioanalyzer and 4-20 % acrylamide gel Group/Presentation Title Agilent Restricted

  42. Detection of Single Base Mutations (2) Group/Presentation Title Agilent Restricted

  43. Label free Analysis of Microsatellite InstabilitiesClinical Diagnostics and Molecular Diagnostics of Cancer • Microsatellite instabilities present in 10-15% of colon and gastric carcinomas • Study: 40 cases of colon carcinoma • 5 microsatellite loci investigated • Results compared with traditional PAGE: • 95% concordance rate Group/Presentation Title Agilent Restricted

  44. Cell Applications Apoptosis Detection Protein Expression Monitoring Intracellular Gene Silencing Protein Expression Monitoring Cell Surface Antibody Staining Transfection Efficiency Monitoring Group/Presentation Title Agilent Restricted

  45. Cell Assay • Apoptosis • Transfection Efficiency Monitoring • Detection of GFP-transfected cells • Antibody staining: Detection of transfected cells expressing the encoded protein • Protein Expression Monitoring • Extracellular and Intracellular Antibody staining for detection of protein expressed on the cell surface, in the cytoplasm, or in the nucleus • Gene silencing Group/Presentation Title Agilent Restricted

  46. Principle of Pressure-Driven FlowFor cell assays (analysis of cell fluorescence parameters)On-chip simple flow cytometric studies Cells pass the fluo-rescence detector in single file and each event is monitored in a histogram or dot plot Pressure driven flow is used to move cells in a controlled manner through the micro-channels Cells are hydrodynamically focused to a portion of the channel by a side stream of buffer The micro-channels of the glass chip are filled with cell buffer Group/Presentation Title Agilent Restricted

  47. The Bioanalyzer Lab-on-a-Chip Approach • Separation on disposable, µ-fabricated glass chips • made of two glass layers: • one with micro-channels (x10µm, etched), • one with through-holes • glued into a plastic caddy which accommodates wells for gel, sample, standard (ladder), buffer and other reagents • for handling nl-amounts of liquids • one separation channel for ladder and sample • microfluidic sample movement with fluorescence detection • Setup • micro-channels are filled with gel or buffer • sample, ladder and reagents are filled into the respective wells • chip preparation in less than 5 minutes • Benefits • convenient handling • minimized risk of cross-contamination • versatile design for multiple experiments on one platform Group/Presentation Title Agilent Restricted

  48. Flow Cytometry on a Chip - Hydrodynamic Focusing • All six cell samples arehydrodynamicly focused to one side of the micro channel • At each of the six pinch points the cell stream is joined by a buffer stream from one of the two buffer wells • The two liquids do not mix immediately • The cells then move towards the detector in single file Group/Presentation Title Agilent Restricted

  49. Flow Cytometry on a Chip - Two Color Detection- Three Types of Events Blue cells Red cells Blue/red cells Dot plot view for easy data evaluation Group/Presentation Title Agilent Restricted

  50. Some Target applications • Apoptosis: Annexin V Detection of phosphatidylserine on the cell surface Caspase-3 Detection of activated caspase-3 in the cytoplasm • Transfection Efficiency Monitoring: GFP: Detection of GFP-transfected cells Antibody staining: Detection of transfected cells expressing the encoded protein • Protein Expression Monitoring: Extracellular and Intracellular Antibody staining for detection of protein expressed on the cell surface, in the cytoplasm, or in the nucleus • Gene silencing: Optimization of siRNA transfection procedure Verify silencing by cellular protein expression measurement Correlation of siRNA uptake and gene knockdown Group/Presentation Title Agilent Restricted

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