iman kishawi, phd senior application scientist

2008

2100 Bioanalyzer

Iman Kishawi, PhDSenior Application Scientist

2008

The Lab-on-a-Chip Approach

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

Increasing quality and speed of gel electrophoresis

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Bioanalyzer Overview

November 2007

Current 2100 Analysis Kits

DNA Assays:1000, 7500, 12000• Sizing

• Quantitation

• PCR products, digests,

larger DNA fragments

• 12 samples in 30 min.

Cell Assays:Flexible use

• Analysis of 6 samples • Two color detection• Analysis of protein expression

in cells

Protein Assays:P80, P230, HSP-250• Sizing

• Quantitation

• cell lysates, column fractions,

purified proteins, antibodies etc.


RNA Assays:nano, pico, Small RNA

• Quantitation (Sizing in Small RNA)

• total RNA, mRNA

• purity & integrity determination


Electrophoretic Separations

Flow Cytometry

2008

Cell Applications

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2008

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

2008

Principle of Pressure-Driven FlowFor cell assays (analysis of cell fluorescence parameters)On-chip simple flow cytometric studies

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The Bioanalyzer Lab-on-a-Chip Approach

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Flow cytometry on the chip - Detection Animation

2008

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

2008

Flow Cytometry on a Chip - Two Color Detection- Three Types of Events

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Some Target applications

Apoptosis:Annexin V Detection of phosphatidylserine on the cell surfaceCaspase-3 Detection of activated caspase-3 in the cytoplasm

Transfection Efficiency Monitoring:GFP: Detection of GFP-transfected cellsAntibody staining: Detection of transfected cells expressing the encoded

proteinProtein 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 procedureVerify silencing by cellular protein expression measurementCorrelation of siRNA uptake and gene knockdown

2008

Typical workflow:

Cell assays: sample preparation

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2100 Bioanalyzer

Red detection channel:

• 620-645 nm excitation with Laser (Maximum 630 nm)

• 674-696 nm detection range (Maximum 680 nm)

Blue detection channel:

• 458-482 nm excitation with LED (Maximum 470 nm)

• 510-540 nm detection range (Maximum 525 nm)

Flow Cytometry on a Chip - Optics & Detection

2008

Cell Assays - Applications: ApoptosisAnnexin Binding

PhosphatidylPhosphatidyl--serine from inner leaflet flips to outer membrane serine from inner leaflet flips to outer membrane during apoptosis and can be labeled by Annexin Vduring apoptosis and can be labeled by Annexin V

Healthy cellHealthy cell

““LiveLive”” apoptotic cellapoptotic cell

Dead cellDead cell

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Annexin V Assay (24h Induction)

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Applications: Protein Expression AnalysisGFP Transfection Efficiency Control

CHO-K1 cells were transfected with EGFP DNA and Lipofectamine.

GFP transfected cells

Mock transfected cells

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2008

GFP Transfection Efficiency

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2100-1 2100-2 2100-3 All Flow cyt.ctrl mean 0.46 0.31 0.47 0.40 0.16

SD 0.08 0.29 0.43 0.29 0.12GFP mean 60.19 59.15 59.26 59.53 60.90

SD 2.13 2.48 2.10 2.26 1.22%CV 3.54 4.19 3.54 3.80 2.01

2008

Flow Cytometry Assays Applications - Cell surface Antibody staining

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Extracellular Antibody Staining

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Mean % CD3+ cells2100-1 2100-2 2100-3 2100-4 Flow cyt.60.9 67.8 66.6 65.0 60.934.4 36.7 36.7 34.3 29.817.3 17.6 18.7 17.2 13.88.9 9.4 9.9 8.3 6.55.1 4.4 5.3 4.9 3.20.8 0.6 0.3 0.3 0.0

2008

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GFP On-Chip Staining - Workflow

2008

GFP On-Chip Staining - Histogram Quality

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Lab-on-a-Chip - Principle of Injection & Separation

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DNA Applications

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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

2008

DNA Kit Specification

2008

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Data Format - Gel-Like Image c/w Agarose Gel

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Determination of PCR Product Impurity

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GMO Detection: Determination of GM Soya Percentage

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Optimization of Multiplex PCR on a 19-plex PCR

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Tumor Diagnostics

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Detection of Single Base Mutations (1)��6��=��"��&��.7�;��!��A+��>�� 6.88�3��

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Detection of Single Base Mutations (2)

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91 bp

91 bp91+83 bp

168 bp

wt 59

276 bp276 bp

251 bp

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p53Exon 7 wt/HpaII p53Exon 7 clone 59/HpaII

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166 bp

84/85 bp

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Time (seconds)

208 bp111 bp

90 bp

2008

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

2008

RNA Applications

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Agilent 2100 bioanalyzer:the industry standard in RNA QC

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

January 2008: ~ 7200 citations

Electrophoretic sizing, quantitation and QC of XNA and Proteins on a small glas Chip as done traditionally on slab gels (Agarose or SDS-PAGE)

Number of BioA publications/month

RIN = RNA Integrity Number, an Agilent patented algorithm to Determine RNA quality in a normalized way

2008

RNA Kit Specifications

2008

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)

RNA integrity 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

Problem Description

The ratio of ribosomal bands is not sufficient to describe RNA integrity!

RNA degradation is a gradual process.

Results have to be interpreted by visual inspection.

Overlay of electropherograms only works well for samples with the same concentration.

Instrument dependency in signal height

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High quality total RNA

Partially degraded total RNA

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RNA Quality Control:Assessing Total RNA Integrity

RIN 9.2

RIN 5.8

2008

Gel Chip Comparison

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RNA QC in Routine Gene Expression Workflow

Start again with sample isolation

Cells / Culture

RNA isolation

Total RNA

RIN

RNA QC via Agilent 2100 bioanalyzer

RIN above threshold

Continue with downstream Experiment (Microarray, real-time PCR, etc.)

2008

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cRNA Hybridization - Workflow

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Incorporation of Cy Dye

Yield of cRNAIntact Partial

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Partial Degradation

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Complete Degradation

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Intact

RIN = 7.3

- Intact (RIN = 7.3)- Partial Degradation (RIN = 5.2)- Complete Degradation (RIN = 2.4)

Scatter Plots : Self vs. Self

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Intact

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RIN = 7.3

Intact vs. Partial Degradation

- Intact (RIN = 7.3)- Partial Degradation (RIN = 5.2)- Complete Degradation (RIN = 2.4)

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Intact vs. Complete Degradation

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Conclusions

� Labeled cRNA from different levels of RNA degradation results in low Cy Dye incorporation and low yield of cRNA

� RNA integrity levels of starting material had serious impact on downstream gene expression microarray results

� The RIN is an effective tool that can be used to evaluate RNA integrity objectively

2008

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rRNA contamination: 19.5 %

rRNA contamination: 1.7 %

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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

2008

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Small RNA Kit

RNA 6000Nano

Size range: 25-6000nt

Results: Integrity, Total RNA amount, gDNA contamination

NEW! Small RNASize range: 6-150nt

Results: miRNA amount, Ratio and amount of other Small RNA

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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

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Intact Total RNA sample

(size separation and relative amount estimation)

Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp

2008

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

2008

Protein Applications

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Bioanalyzer Protein Kit portfolio

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P 80

Range 5 - 80 kDaSensitivity: CoomassieSamples: 10

Samples-Antibodies (reduced)-Small Proteins

P 230

Range 14 - 230 kDaSensitivity: CoomassieSamples: 10

Samples-Antibodies (all types)-Standard Proteins

Range: 10 - 250 kDaSensitivity: 1 pg/µl BSA on ChipSamples #: 10 per ChipChips #: 10 per KitLabeling Conc: 1 ng – 1 µg /µl

Coomassie Range (5 ng/µL BSA)

HSP 250

Silver stain Range (200 pg/µL BSA)

Agilent Protein 80 kit Prod Number 5067-1515Agilent Protein 230 kit Prod Number 5067-1517Agilent High Sensitivity Protein 250 kit Prod Number 5067-1575

2008

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.

Product No. 5067-1515 Product No. 5067-1517 Product No. 5067-1575

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Clone Selection based on Protein Expression

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2008

Monitoring of Protein Purification Process

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Example measured withProtein 200 plus kit

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Expression of a Recombinant Protein in E.coli- Optimization of Fermentation and Induction Conditions

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recombinant protein

soluble protein fraction

solubilized inclusion bodies (50 mM Tris pH 7.5, 100 mM DTT, 8M urea)

2008

Quality control of the Depletion of High Abundance Proteins in Human Serum

Depletion of High Abundance Proteins by Agilent MARS HPLC columns

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2008

Quality Control of Antibodies

90 kDa 160 kDa

intact antibody

16% half antibody

Determine the half antibody content in IgG preparations

Antibody analysis under reducing and non-reducing conditions

Ab reduced

Ab non-reduced

light chain

heavy chain

intact antibody

light + heavy chain

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Ab non-reduced

Absolute Quantitation of IgG samples

2008

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Antibody 1- Standard Antibody 1- 1 month 40°C

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Antibody 2- Standard Antibody 2- 12 weeks 40°C

Analysis of Antibody Stability – stress test

2008

Combination of IEF with SDS-PAGEAgilent 3100 OFFGel Fractionator + 2100 bioanalyzer

2008

Description of the new HSP-250 Assay(Direct labeling reaction, silver stain sensitivity)

• Reach and beat traditional „silver stain sensitivity“

• Offer solid quantitation for a large dynamic range

Target Applications:• Protein QA/QC

reliable quantitation of main compound besides minor impurities

• Protein detection at lowest concentrations in research

High Sensitivity Protein 250 Kit 5067-1575 content is:- 10 Chips (100 samples)- Labeling Kit (Dye and Reagents)- 2100 Separation Kit (Gel, Marker, Ladder, Buffer)- User Documentation (Quick Start Guide & Labeling Protocol)

2008

Extended experimental workflow

Transfer to suitable buffer(precipitation, ultrafiltration, buffer exchange spin columns)

Labeling with dye(N-hydroxy-succinimidyl ester chemistry)

Sample preparation for 2100(SDS denaturation, dilution if desired)

Analysis on 2100

labeling kit

separation kit

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40 min

5 min

35 min

Sample

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2008

Principle of High Sensitivity 250 Protein Staining

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2008

Reproducibility of Labeling Reaction: Ladder

Rugged Labeling reaction:Reproducible reaction provides comparable signal intensities. Homogenous labeling without extra bandbroadening

No deviation in peak width is indicating a constant number of dye per protein molecule and proves a stable protocol

2008

Sensitivity: Silver Staining vs. Bioanalyzer

Highest sensitivity:Labeled proteins can be measured down to pg/µL concentrations loaded on Chip

Direct comparison of samples run on SDS-PAGE with Silver staining and on 2100 Bioanalyzer.

Concentrations are given per lane (as total concentration of 7 different proteins)

2008

Linear Dynamic Range Test: IgG

Linear dynamic range:Quantification of labeled IgG from 10 pg/µL to 100 ng/µLaverages ± SD of 7 measurements (7 chips, 1 chip lots, 4 instruments)

4 orders of linear dynamic range allows to quantify an 0.05% impurity besides the main peak in a single run

2008

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

2008

Agilent Web pages with 2100 content

www.opengenomics.com

www.agilent.com/chem/labonachip

2008

2100 kits for Protein applications

Number Kit Max # of samples

5067-1517 Agilent Protein 230 Kit 250

5067-1518 Agilent Protein 230 Reagents

5067-1515 Agilent Protein 80 Kit 250

5067-1516 Agilent Protein 80 Reagents

Protein Kits – Coomassie stain sensitivity


5067-1575 High sensitivity Protein 250 Kit 100

5067-1576 High sensitivity Protein 250 Reagents

5067-1577 High sensitivity Protein 250 Labeling Reagents

5067-1578 High sensitivity Protein 250 Ladder

Protein Kits – Silver stain Sensitivity

2008

2100 kits for Cell Assay and DNA applications


5067-1519 Agilent Cell Kit 150

5067-1520 Cell Checkout Kit

Cell Fluorescence Kits


5067-1504 Agilent DNA 1000 Kit 300

5067-1505 Agilent DNA 1000 Reagents

5067-1506 Agilent DNA 7500 Kit 300


5067-1508 Agilent DNA 12000 Kit 300


DNA Kits

2008

2100 kits for RNA applications


5067-1511 Agilent RNA 6000 Nano Kit 300

5067-1512 Agilent RNA 6000 Nano Reagents

5067-1529 Agilent RNA 6000 Nano Ladder

5067-1513 Agilent RNA 6000 Pico Kit 275

5067-1514 Agilent RNA 6000 Nano Reagents

5067-1535 Agilent RNA 6000 Nano Ladder

5067-1548 Agilent Small RNA Kit 275

5067-1549 Agilent Small RNA Reagents

5067-1550 Agilent Small RNA Ladder

RNA Kits

2008

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Time (seconds)

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25 30 35 40 45 50 55 60 65 70 75 80 85 90

3000 bp PCR 3000 bp PCR 1:4 dil.

Impurity level : > 50%3000 bp

Determination of PCR Product Impurity1* 2 3 4*

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Time (seconds)

0

100

200

300

400

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25 30 35 40 45 50 55 60 65 70 75 80 85 90

300 bp PCR 300 bp PCR 1:4 dil.

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300 bp

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iman kishawi, phd senior application scientist

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