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2100 Bioanalyzer

2100 Bioanalyzer . Assay Portfolio Overview July 2008. The Lab-on-a-Chip Approach. Increasing quality and speed of gel electrophoresis. Sample volumes 1 - 5 µl 10 -12 samples depending on Assay Separation, staining, detection of samples Results in 5-30 minutes available

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2100 Bioanalyzer

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  1. 2100 Bioanalyzer Assay Portfolio Overview July 2008

  2. The Lab-on-a-Chip Approach Increasing quality and speed of gel electrophoresis Sample volumes 1 - 5 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

  3. Lab-on-a-Chip or Gel - A Faster Answer !

  4. Electrophoretic Separations Flow Cytometry Current 2100 Analysis Kits • DNA Assays: • 1000, 7500, 12000 • Sizing • Quantitation • PCR products, digests, larger DNA fragments • 12 samples in 30 min. • RNA Assays:nano, pico, Small RNA • Quantitation (Sizing in Small RNA) • total RNA, mRNA • purity & integrity determination • 10 samples in 30 min. • Protein Assays: • P80, P230, HSP-250 • Sizing • Quantitation • cell lysates, column fractions, purified proteins, antibodies etc. • 10 samples in 40 min. Cell Assays: Flexible use •Analysis of 6 samples •Two color detection • Analysis of protein expression in cells Bioanalyzer Overview Page 4 November 2007

  5. 2100 kits for Protein applications Protein Kits – Coomassie stain sensitivity Protein Kits – Silver stain Sensitivity

  6. 2100 kits for Cell Assay and DNA applications Cell Fluorescence Kits DNA Kits

  7. 2100 kits for RNA applications RNA Kits

  8. RNA Applications RNA QA/QC for Microarrays Gene Expression RNA QA/QC for qPCR RNA QA/QC for mPCR smallRNA QA/QC

  9. Agilent 2100 bioanalyzer: the industry standard in RNA QC Electrophoretic sizing, quantitation and QC of XNA and Proteins on a small glas Chip as done traditionally on slab gels (Agarose or SDS-PAGE) First commercially available Lab-on-a-Chip product (since October 1999) Analysis of totalRNA, mRNA and small RNA samples in ng and pg concentration range Standardized RNA integrity assessment with RIN*algorithm Multi-analysis capabilities: DNA, RNA, Proteins and Flow Cytometry Number of BioA publications/month RIN = RNA Integrity Number, an Agilent patented algorithm to Determine RNA quality in a normalized way January 2008: ~ 7200 citations

  10. RNA Kit Specifications

  11. 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

  12. RNA Quality Control: Assessing Total RNA Integrity 11 18S 28S 10 9 8 7 marker 6 Fluorescence 5 4 3 2 1 0 28S 18S 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) Typical first QC step after RNA sample prep prior to microarrays or real-time PCR RIN 9.2 High quality total RNA RIN 5.8 Partially degraded total RNA

  13. Gel Chip Comparison False Negative False Positive Data kindly provided by Gene Logic Inc. Data kindly provided by Gene Logic Inc.

  14. 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.)

  15. 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

  16. 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

  17. 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 %

  18. 200 pg total RNA 18S 28S 24 29 34 39 44 49 54 59 64 69 Laser Microdissection – PALM MicroBeam System and RNA Pico kit 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) Data kindly provided by P.A.L.M. Microlaser Technologies

  19. Cy3/ Cy5 Labeling mRNA QC Total RNA QC cRNA fragmentation Data evaluation cRNA Hybridization - Workflow Array experiment

  20. qPCR - Why Quality Matters RNA quality control (Quality of template): • RNA degrades naturally due to enzymatic or autocatalytic mechanisms:  Any 5’ or 3’ biased design might fail or produce misleading results  Wrong priming strategy in the RT step can produce misleading results • Knowing RNA quality allows to accommodate the amplicon design and set expectations avoiding wrong interpretatio of results • All quantifications rely on comparable template quality to be meaningful Sample/templatequality

  21. qPCR - Why Quality Matters Sample/templatequality • QPCR assay validation/optimization (Quality of results): • The resolution of SYBR Green meltcurves is limited • Tm depends on dye/template ratio • SYBR Green is a non-saturating dye • Verifying the size of PCR products is a recommended validation procedure:  Resolution of slab gels limited! Quality of assay Robust and meaningfulresults

  22. Mx3005P® qPCR quality control - Experimental workflow Extraction from 5x106HEK cells usingAbsolutely RNA® mini RNA extraction • Quantification of 1 µl sample on Nanodrop • QC on Bioanalyzer:RNA 6000 nano kit Nucleic acid quantification and QC RNA degradation @ 70°C RT from 1 µg of totalRNA usingAffinityScript™ Reverse Transcription QPCR 5‘ and 3‘ assays QPCR Assay validation Analysis of QPCR products onBioanalyzer: DNA 1000 kit Brilliant® II SYBR® Green

  23. oligo-dT random Size: 110 bp oligo-dT random Size: 21 + 51 bp NTC Size: 121 bp Ensuring Quality of Results Assay Validation - Specificity Benefit from the superior resolution of the Bioanalyzer: A Validation of amplicon size:GAPDH amplicons (A) 118 bp(B) 126 bp B Validation of unclear results

  24. HPRT1 GAPDH YWHAZ A B RIN 8.90 min RIN 6.5 30 min C D RIN 4.6 45 min RIN 2.3 75 min Quality and Impact on gene expression results • Effect of RNA quality ongene expression results: • RNA was extracted from HEK293 cells and thermally degraded • All RNAs were tested on the Agilent Bioanalyzer Assay design: Results: GAPDH HPRT1 YWHAZ intron 2-3: 23.6 kb

  25. 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

  26. 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

  27. Intact Total RNA sample LM rRNA rRNA tRNA miRNA ApplicationsThe new small RNA Assay as a tool for: • Verification, comparison and optimization in the small RNA region: • High sensitivity to detect low abundant fragments • High resolution for ss oligos, miRNA, pre-, t-, 5S-RNA’s • compatible with Total RNA samples or purified small RNAs. • Semi-quantitative for single stranded RNA. • semi- Denaturing • Analysis up to 150nt Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp (size separation and relative amount estimation)

  28. 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

  29. DNA Applications mPCR validation, impurity check Gene Expression Restriction Digest Analysis Food Analysis Forensic Testing

  30. 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

  31. DNA Kit Specification

  32. 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

  33. 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

  34. 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%

  35. 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

  36. Optimization of Multiplex PCR on a 19-plex PCR Data kindly provided by QIAGEN GmbH, Germany

  37. 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

  38. 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

  39. Detection of Single Base Mutations (2)

  40. 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

  41. Protein Applications Protein Purification Protein Expression Protein Production Food Analysis Purity and QA/QC

  42. Agilent Protein 80 kit Prod Number 5067-1515 Agilent Protein 230 kit Prod Number 5067-1517 Agilent High Sensitivity Protein 250 kit Prod Number 5067-1575 Bioanalyzer Protein Kit portfolio Introduction HSP 250 • P 80 • Range 5 - 80 kDa • Sensitivity: Coomassie • Samples: 10 • Samples • Antibodies (reduced) • Small Proteins • P 230 • Range 14 - 230 kDa • Sensitivity: Coomassie • Samples: 10 • Samples • Antibodies (all types) • Standard Proteins Range: 10 - 250 kDa Sensitivity: 1 pg/µl BSA on Chip Samples #: 10 per Chip Chips #: 10 per Kit Labeling Conc: 1 ng – 1 µg /µl Coomassie Range (5 ng/µL BSA) Silver stain Range (200 pg/µL BSA)

  43. New Product No. 5067-1515 Product No. 5067-1517 Product No. 5067-1575 * Protein Kit Specifications *Prior to measurement on Chip we recommend within the High Sensitivity Protein 250 labeling protocol to dilute the labeled sample by a factor of 200.

  44. Avoiding high background, providing better Sensitivity SDS + dye If no dilution was done the micelles would result in high background and low sensitivity protein micelles destain detection low background good signal to noise ratio SDS conc. below CMC Staining, Destaining and Detection Staining, Destaining and Detection (P-80 and P-230)

  45. 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)

  46. 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

  47. soluble protein fraction recombinant protein solubilized inclusion bodies (50 mM Tris pH 7.5, 100 mM DTT, 8M urea) Expression of a Recombinant Protein in E.coli- Optimization of Fermentation and Induction Conditions

  48. Depletion of High Abundance Proteins by Agilent MARS HPLC columns A B Fractions checked by 2100 Quality control of the Depletion of High Abundance Proteins in Human Serum

  49. 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

  50. A B Antibody 1- Standard Antibody 1- 1 month 40°C Antibody 1 – 1 month 40°C Antibody 2 – 12 weeks 40°C 600 Antibody 2 - Standard 500 400 Fluorescence 300 200 210 100 0 21 23 25 27 29 31 33 35 Time (seconds) 117.0 Antibody 2- Standard Antibody 2- 12 weeks 40°C 97.4 600 66.7 HC 53.0 500 32.5 400 29.0 Fluorescence LC 300 21.5 14.4 200 100 LM 0 21 23 25 27 29 31 33 35 Time (seconds) Analysis of Antibody Stability – stress test Ladder Antibody 1 - Standard

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