Agilent 2100 Bioanalzyer Applications Master Set

Page 120082100 Bioanalyzer

Assay Portfolio OverviewJuly 2008

Page 22008The Lab-on-a-Chip Approach

Sample volumes 1 - 5 l10 -12 samples depending on AssaySeparation, staining, detection of samplesResults in 5-30 minutes availableNo extra waste removal neededDisposable Chip, no crosscontamination

Increasing quality and speed of gel electrophoresis2This slide shows such a microfluidic chip.The chip for protein analysis consists, like all other bioanalyzer chips, of 2 glass layers bond together by high temperature and high pressure. One glass layer has micro-fabricated channels etched into it. The other layer closes the channels and has through-holes that form the sample and buffer reservoirs and provide access to the channels.The chips do not contain any mechanical parts. Using microfluidic technology allows to actively control fluids on the chip without any moving parts.The protein chip for example, contains the emulation of pumps, valves and dispensers for sample handling on the chip, and a separation system to electrophoretically separate the proteins according to their size. The protein chip also has a system that allows the staining and destaining process to be integrated onto the chip. The protein chip offers a higher degree of functionality compared to the other DNA and RNA chips, bringing us one step closer to an actual laboratory-on-a-chip.

Separation on disposable, -fabricated glass chipsmade of two glass layers: one with micro-channels (x10m, etched), one with through-holesglued into a plastic caddy which accommodates wells for gel, sample, standard (ladder), buffer and other reagents for handling nl-amounts of liquidsone separation channel for ladder and samplemicrofluidic sample movement with fluorescence detectionSetupmicro-channels are filled with gel or buffersample, ladder and reagents are filled into the respective wellschip preparation in less than 5 minutesBenefitsconvenient handlingminimized risk of cross-contaminationversatile design for multiple experiments on one platform

Read!Chip priming (with gel or buffer) and sample loading onto the chip is the only manual interaction which is needed prior to the run. After the chip is placed in the bioanalyzer everything runs automaticallly - sample handling, separation, detection and analysis.Page 32008Lab-on-a-Chip or Gel - A Faster Answer !

3This slide shows a time comparison of the workflow for the bioanalyzer and conventional gel electrophoresis. The sample preparation is comparable for both techniques. Once the samples are loaded on to the chip analysis is completed within 30 minutes on the bioanalyzer. No additional manual steps, such staining/destaining and data analysis are needed. The data is provided in a clean workable form, all analyzed and archived within 40 minutes.To run a precast protein gel, the same sample preparation and setup time is required. The next steps already makes the differences in comparison to the bioanalyzer. To run a gel takes approximately 30 minutes to 2 hours depending on the type of gel. This is followed by staining and destaining steps. The time for staining/destaining can vary significantly depending on the staining protocol used. Some people do a quick staining, followed by a destaining step in the microwave, but often staining and destaining needs to be done overnight to obtain optimal results.To get exactly the same level of information as with the bioanalyzer, the gel then needs to be scanned and analyzed, which also takes a considerable amount of time.All in all this adds up to a minimum of 2-3 hours compared to 45 minutes from the bioanalyzer.

Page 42008Page 4Bioanalyzer Overview

November 2007Current 2100 Analysis KitsDNA Assays:1000, 7500, 12000SizingQuantitationPCR products, digests, larger DNA fragments12 samples in 30 min.Cell Assays:Flexible use

Analysis of 6 samples Two color detection Analysis of protein expression in cellsProtein Assays:P80, P230, HSP-250SizingQuantitationcell lysates, column fractions, purified proteins, antibodies etc.10 samples in 40 min.

RNA Assays:nano, pico, Small RNA Quantitation (Sizing in Small RNA) total RNA, mRNA purity & integrity determination 10 samples in 30 min.Electrophoretic SeparationsFlow Cytometry

44Page 520082100 kits for Protein applicationsNumberKitMax # of samples5067-1517Agilent Protein 230 Kit2505067-1518Agilent Protein 230 Reagents5067-1515Agilent Protein 80 Kit 2505067-1516Agilent Protein 80 Reagents Protein Kits Coomassie stain sensitivityNumberKitMax # of samples5067-1575High sensitivity Protein 250 Kit1005067-1576High sensitivity Protein 250 Reagents5067-1577High sensitivity Protein 250 Labeling Reagents5067-1578High sensitivity Protein 250 Ladder Protein Kits Silver stain SensitivityPage 620082100 kits for Cell Assay and DNA applicationsNumberKitMax # of samples5067-1519Agilent Cell Kit 1505067-1520Cell Checkout KitCell Fluorescence Kits NumberKitMax # of samples5067-1504Agilent DNA 1000 Kit3005067-1505Agilent DNA 1000 Reagents5067-1506Agilent DNA 7500 Kit3005067-1507Agilent DNA 7500 Reagents5067-1508Agilent DNA 12000 Kit3005067-1509Agilent DNA 12000 ReagentsDNA Kits Page 720082100 kits for RNA applicationsNumberKitMax # of samples5067-1511Agilent RNA 6000 Nano Kit3005067-1512Agilent RNA 6000 Nano Reagents5067-1529Agilent RNA 6000 Nano Ladder 5067-1513Agilent RNA 6000 Pico Kit2755067-1514Agilent RNA 6000 Nano Reagents5067-1535Agilent RNA 6000 Nano Ladder 5067-1548Agilent Small RNA Kit2755067-1549Agilent Small RNA Reagents5067-1550Agilent Small RNA Ladder RNA Kits Page 82008RNA ApplicationsRNA QA/QC for MicroarraysGene ExpressionRNA QA/QC for qPCRRNA QA/QC for mPCRsmallRNA QA/QC

Page 92008Agilent 2100 bioanalyzer:the industry standard in RNA QCFirst commercially available Lab-on-a-Chip product (since October 1999)Analysis of totalRNA, mRNA and small RNA samples in ng and pg concentration rangeStandardized RNA integrity assessment with RIN* algorithmMulti-analysis capabilities: DNA, RNA, Proteins and Flow CytometryJanuary 2008: ~ 7200 citationsElectrophoretic 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 way9Page 102008RNA Kit Specifications

10Specification of the RNA 6000 Nano kitPage 112008total RNA determine integrity and quality of total RNAdetermination of RNA concentrationidentify ribosomal peaks calculate the ratio of ribosomal peaks (18S/28S or 16S/23S)RNA integrity number (RIN)

mRNA determine integrity and quality of mRNA samplesDetermination of mRNA concentration calculate % ribosomal RNA in mRNA samples

Features of the RNA 6000 Assays

11The table lists areas of use for 2100 bioanalyzer.Page 122008High quality total RNAPartially degraded total RNA18S28SFluorescenceTime (seconds)01234567891011 19 24 29 34 39 44 49 54 59 64 69marker18S28SFluorescenceTime (seconds)01234567891011 19 24 29 34 39 44 49 54 59 64 69marker

28S18STypical first QC step after RNA sample prep prior to microarrays or real-time PCRRNA Quality Control:Assessing Total RNA Integrity RIN 9.2RIN 5.812This slide shows the analysis of total RNA integrity - a typical first QC step during cDNA or cRNA sample prep for microarrays.The upper eleectropherogram and gel-like image show the analysis of high quality total RNA with the 18S and 28S subunit clearly distinctible.The lower part of the slide shows an e-gram and gal-like image typically resulting from th analysis of partially degraded total RNA. No distinct peaks but only a hump or smear is detected.With the help of the 2100 bioanalyzer and the RNA Nano kit the important sample QC step prior to an expensive microarray experiment can be easily and quickly achieved.Page 132008Gel Chip Comparison

False NegativeData kindly provided by Gene Logic Inc.

False PositiveData kindly provided by Gene Logic Inc.13False NegativePart A) shows the band absence in the lane marked with an arrow, yet the two bands are clearly seen as peaks in the electropherogram in B). The 28S/18S ratio for this sample is 1.09.Sample DegradationPart A) marked with an arrow shows a missing ribosomal band, while Part B) shows both the 18S and 28S bands present (note that some degradation is present, therefore additional Quality Control measures may be needed). The 28S/18S ratio for this sample is 0.90.

Page 142008RNA QC in Routine Gene Expression WorkflowStart again with sample isolation

Cells / CultureRNA isolationTotal RNARINRNA QC via Agilent 2100 bioanalyzerRIN above thresholdContinue with downstream Experiment (Microarray, real-time PCR, etc.)14Page 152008108 samples 3 dilutionsCV RIN: 3 %CV ribosomal ratio: 22 %RIN Application Directly Compare Samplessame sample in different dilutions18S28SFluorescence0.02.55.07.510.012.518S28SFluorescenceTime (seconds)01020304050607080 19 24 29 34 39 44 49 54 59 64 6918S28SFluorescence05101520 25 ng/l: RIN 8100 ng/l: RIN 8500 ng/l: RIN 8When testing an identical RNA sample in various dilutions, identical RINs are obtained within narrow limits15Page 162008RIN Application Assessment of RNA IntegrityIntact RNA: RIN 10

Partially degraded RNA: RIN 5

Strongly Degraded RNA: RIN 3

18S28SFluorescence012345678918S28SFluorescence051015202530354045FluorescenceTime (seconds)0.00.51.01.52.02.53.03.5 19 24 29 34 39 44 49 54 59 64 6916Page 172008

rRNA contamination: 19.5 %rRNA contamination: 1.7 %

Mouse kidney I mRNAMouse kidney II mRNARat brain mRNABovine kidney mRNAMouse testis mRNAMouse liver mRNARNA 6000 ladderRibosomal RNA contamination in mRNA samples17During the isolation of mRNA, varying amounts of ribosomal RNA can remain in a sample. Since the purity of mRNA is of importance for a number of downstream applications, samples should be checked on the bioanalyzer. The current slide shows the analysis of 6 commercially available RNA samples from different suppliers. Analysis on the bioanalyzer reveals large differences in the purity of the mRNA samples.Page 182008Laser Microdissection and Pressure Catapulting (LMPC)RNA extractionABCRNA sample QC using the Agilent 2100 bioanalyzer and the RNA 6000 Pico LabChip kitLaser Microdissection PALM MicroBeam System and RNA Pico kitData kindly provided by P.A.L.M. Microlaser Technologies Laser microdissectionLaser pressure catapulting: Section after catapulting of selected areaCatapulted area in the collection device18S28S 24 29 34 39 44 49 54 59 64 69200 pg total RNA

18Page 192008

cRNA fragmentation

Cy3/ Cy5 Labeling

Total RNA QC

mRNA QCArray experimentcRNA Hybridization - Workflow

Data evaluation19Examples of where the bioanalyzer can be used in the microarray workflow:Isolation of total RNA (QC and quantitation).Isolation of mRNA (QC and quantitation).Detection of Cy5 (Cy3) labeled samples (QC and dye incorporation for CY5).cRNA fragmentationMicroarray experiments that are performed with high quality samples have a high chance of resulting in high quality data.

Page 202008qPCR - Why Quality MattersSample/templatequalityRNA 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 meaningful20High quality template will produce high quality and meaningful results. RNA and DNA both degrade through various mechanisms. In gene expression analysis wrong priming strategy and/or wrong positioning of the amplicon can lead to failure or over-/underestimation of the real quantity/fold-change (in genotyping or pathogen detection the same applies to copynumber, quantity etc)

Page 212008qPCR - Why Quality MattersSample/templatequalityQuality of assayRobust and meaningfulresultsQPCR 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!21As proper validation is also recommended to ensure quality of results, verification of specificity should be performed with every new assay.SYBR Green melt curve analysis has some limitations and wont provide you with the size of the amplicon. Due to these limitations even the same amplicon can give rise to different Tm values.

Page 222008qPCR quality control - Experimental workflowRNA extractionNucleic acid quantificationand QCReverse TranscriptionQPCR Assay validationQPCR 5 and 3 assaysExtraction from 5x106HEK cells usingAbsolutely RNA miniRT from 1 g of totalRNA usingAffinityScript Quantification of 1 l sample on Nanodrop QC on Bioanalyzer: RNA 6000 nano kitAnalysis of QPCR products onBioanalyzer: DNA 1000 kitRNA degradation @ 70C

Mx3005PBrilliant II SYBR Green22The experiments done used RNA purified from cultured HEK cells with Absolutely RNA mini kit. The RNA was quantified using Nanodrop and quantification/QC was done using the Agilent Bioanalyzer.RNA was reverse transcribed with AffyityScript and the resulting cDNA used for assay validation and for the degradation experiments.Assay validation was performed on the Mx using Brilliant II SYBR with a melt curve analysis and a further validation of PCR product size on the Bioanalyzer.For the degradation the RNA was incubated at 70C for various times and the degraded RNA was tested on the Bioanalyzer to determine the extend of degradation as indicated by the RIN number. With the resulting cDNA QPCR was performed (again using Brilliant II SYBR) using 5 as well as 3 biased amplicons for several genes. Page 232008

Size: 110 bp

oligo-dTrandom

Size: 121 bp

oligo-dTrandom

NTC

Size: 21 + 51 bpEnsuring Quality of ResultsAssay Validation - SpecificityBenefit from the superior resolution of the Bioanalyzer: Validation of amplicon size:GAPDH amplicons (A) 118 bp(B) 126 bpValidation of unclear resultsAB23Example of the assay validation for GAPDH. For validation two samples derived from human QPCR reference RNA and HEK total RNA were used. All RNAs were reverse transcribed using either oligo-dT priming or random priming to determine the optimal priming strategy with intact RNA. The picture shows the amplifications plots, melt curve analysis as well as Bioanalyzer DNA analysis.Oligo-dT based priming gave earlier Ct values therefore it was used in all subsequent experiments. The deviation of the size calculated by the Bioanalyzer over the size determined from the sequence could be due to the fact that the QPCr reaction including salts and SYBR green was used.

Assay validation showed negative noRT controls for all assays and all samples, so no genomic DNA was present (YWHAZ 3 assay). Almost all NTC controls were negative with the exception of 1 NTC for the HPRT1 5 assay. The meltcurve showed a shallow peak in the NTC that had a Tm very close to the actual product in the cDNA samples. From this it was not clear if this was a contamination or primer dimers. Analysis of the NTC on the Bioanalyzer shows two minor peaks with 21 bp and 51 bp indicating primer dimer formation. This nicely shows that under certain circumstances the increased resolution achieved with the Bioanalyzer for DNA analysis can really make a difference. It also shows that also the size difference between amplicon and primer dimer (167 bp vs. 51 bp) results in just a minor difference in Tm.

Page 242008intron 2-3: 23.6 kbGAPDHHPRT1YWHAZ

RIN 8.90 minARIN 6.530 minB

RIN 4.645 minC

RIN 2.375 minD

GAPDH

HPRT1YWHAZ

Effect of RNA quality ongene expression results:

RNA was extracted from HEK293 cells and thermally degraded All RNAs were tested on the Agilent BioanalyzerQuality and Impact on gene expression resultsResults:Assay design:24Target genes used in the experiment were choosen to be on different chromosomes, having different expression levels and for the fact that they had been used in similar studies.GAPDH/HPRT1: both primer pairs were designed to have at least one primer spanning exon junctionsYWHAZ: Due to sequence restrictions the 5 amplicon primer pair was designed to be in two different exons with a 23.6 kb intron inbetween.The 3 biased amplicon for YWHAZ was deliberatly designed to be all in one exon to detect possible genomic DNA contaminations

To demonstrate the effect of RNA quality on the outcome of QRCR results, RNA was thermally degraded at 70C. The incubation was stopped at 30, 45 and 75 min. In the next step 1 l of the thermally degraded RNA was analyzed using the RNA 6000 Nano assay. The RNA integrity number (RIN) was automatically determined. The 2100 bioanalyzer electropherogram of the intact RNA shows the typical ribosomal peaks, which disappear in the course of degradation. Additionally, a shift to smaller sizes is observed. The corresponding RIN range from 8.9 which indicates good RNA quality to 2.3 (highly degraded).

For each of the RNA samples with RIN from 8.9 to 2.3, QPCR was carried out to quantify the amount 5and 3 for the three tested genes.The relative quantities of the respective cDNA copies towards the RNA sample with RIN 8.9 were calculated.A significant difference in the relative quantities is observed for all samples with RIN 4.6 or 2.3.In addition, for the highly degraded RNA with RIN 2.3, the extend of degradation differs for 5and 3 end seems to be gene specific.The data indicates that the integrity of RNA templates can significantly influence the outcome of a QPCR experiment.

Page 252008Analysis of Small RNA (using RNA 6000 Assay)Small RNA fraction: < 200 nts e.g. miRNA, siRNA, snRNA, tRNA, 5S RNABioanalyzer allows discrimination of different profiles

25Page 262008

Small RNA Region

New Small RNA Assay versus existing RNA Assay

miRNA RegionLower MarkerSmall RNA RegiontRNA18s28sRIN: 8.1Lower MarkerRNA 6000Nano kitSmall RNA Kit

RNA 6000NanoSize range: 25-6000ntResults: Integrity, Total RNA amount, gDNA contaminationNEW! Small RNASize range: 6-150ntResults: miRNA amount, Ratio and amount of other Small RNA5s5.8s26Figure 1. Low expression levels of miRNA in Skeletal Muscle RNA are easily resolved with the new Small RNA kit. Panel A shows the results form the RNA 6000 assay, providing information on total RNA concentration, purity and Integrity. The Small RNA assay running the same sample, let you resolve the small RNA fraction and quantify the

Page 272008ApplicationsThe 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-RNAs compatible with Total RNA samples or purified small RNAs. Semi-quantitative for single stranded RNA. semi- Denaturing Analysis up to 150nt

miRNAtRNArRNArRNALMIntact Total RNA sample(size separation and relative amount estimation) Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp27Denaturation? Conditons as much as we can.Remove aggregation, 40C - Page 282008Small RNA Assay specificationsAnalytical Range 6 -150 nt (to avoid overlap)Sensitivity 50 pg/l (diluted Ladder - 40 nt fragment; S/N > 3:1)Quantitative range50 pg/l 2000 pg/l (purified miRNA in water after extraction ~ 50%3000 bpDetermination of PCR Product Impurity

Impurity level : < 2%300 bpQuantitative data from Agilent 2100 bioanalyzer

Sample c (DNA) main peak3000 bp PCR 61.9 ng/ul 40.7 ng/ul3000 bp PCR 1:4 14.8 ng/ul 9.8 ng/ul

34Can use chip to examine quality of PCR products and the amount of non-specific products in the reaction prior to downstream applications ie. spotting PCR products on microarrays

This slide shows the comparison between the analysis of two PCR reactions (300 and 3000 bp products) using the chip vs. an agarose gelTwo different concentrations are shown side by side for each PCR reaction.The next slide indicates the quantitative data4:1 dilutionthe bioanalyzer does a better job at locating impurities over a broader concentration range than the gel300bp appears to be uncontaminated in both the gel and 21003000bp can barely see the impurity with the gel. Cant see with the 1:4 dilution

The quantitative data generated using the bioanalyzer indicates the amount of impurity or non-specific products in these PCR reactions.The 300bp product is very pure vs. the 3000 bp product had several non-specific bands that can be quantitated. This slide tells how much impurityPage 352008GMO Detection: Determination of GM Soya Percentage

Data kindly provided by CCFRASoya lectin gene targetEPSPS gene target: specific for Roundup Ready GM soya (Monsanto)35Multiplex assay for GM soya. The aim was to develop a model assay that could be used to assess the quality of DNA extracted from heat-processed soya flour samples, in particular, to investigate differences in PCR amplification between small DNA targets. A single multiplex PCR assay was developed that enabled four GM soya targets to be analyzed in a single reaction mix. Primer concentration was optimized in order to obtain four PCR products resolved by gel electrophoresis which corresponded in size to the soya lectin gene target of 80 bp, and the EPSPS (5-enolpyruvyl-shikamate- 3-phosphate synthase) gene targets of 117 bp, 150 bp and 202 bp respectively. These latter targets are only found in Roundup Ready GM soya Peaks produced by the four PCR products when analyzed with the Agilent 2100 bioanalyzer and DNA 500 LabChip kit.

Increasing amounts of Soya in reference materials after multiplex RT-PCR. Normalization to the upper marker helps to minimize run-to-run variations and work under standardized conditions. Unknown samples are then compared to the normalized bioanalyzer data. A good estimation of the amount of GM soya can be obtained corresponding to data obtained by real time PCR (light cycler, TaqMan).Page 362008Optimization of Multiplex PCR on a 19-plex PCR

Data kindly provided by QIAGEN GmbH, Germany

36Page 372008

Tumor DiagnosticsData kindly provided by AdnagenSpiking experiment with given amount of cancer cells Enrichment with AdneGen Cancer Select kit (antibody based immunomagnetic enrichment.)Multiplex Amplification with AdnaGen CancerDetect kitDetection with Agilent 2100 Bioanalyzer and DNA 500 LabChip kit

37Sensitivity of AdnaTest CancerSelect/CancerDetect kit analyzed with the 2100 bioanalyzer (Agilent Technologies). A defined number of 2-10 carcinoma cells were spiked to 5 mL of blood from healthy donors. Followed by tumor cell enrichment with AdnaTest CancerSelect and a multiplex PCR, using the AdnaTest CancerDetect kit.Workflow:Spiking experiment with given amount of cancer cellsEnrichment with AdneGen Cance Select kit Multiplerx Amplification with AdnaGen CancerDetect kitDetection with Agilent 2100 Bioanalyzer and DNA 500 LabChip kit

Page 382008Detection of Single Base Mutations (1)in Exons 7 and 8 of the Human p53 Gene by RFLP Mapping using the DNA 7500 kitAmplify exons 7 and 8 (resulting products: 618 bp fragment and 200 bp fragment)Digest with Hpa IIAnalyze using Agilent 2100 bioanalyzer and 4-20 % acrylamide gelexon 7exon 883 bp, 91 bp,168 bp, 276 bp91 bp, 109 bpIn each example one of the restriction sites can be deleted by a point mutation

38The gels were stained with SYBR Gold (Molecular Probes) and scanned on a Fluorimager 595 (Molecular Dynamics). Mutation detection by RFLP highlights the use of the 2100 bioanalyzer, if high sizing accuracy is requiredExperiment:- Isolation of two different regions of p53 gene by using PCR with appropriate primers- digestion with HpaII (enzyme), which cuts in a location that is prone to mutations- analysis of fragment pattern on slab gel to see if mutation is present (if mutation is present the restriction site is not recognized by the enzyme - and therefore not cut - two smaller fragments disappear, one larger fragment appears).

Page 392008Detection of Single Base Mutations (2)

39Analysis on the LabChip showed an identical pattern of digest fragments as seen via the Slab gel for the wildtype and exon 7 & 8 PCR products.Comparison of the calculated sizes of the bands shows 1-2% variance with the Lab Chip system.Remember that LabChip takes 90sec, Slab gel takes 1hr +

Page 402008Label free Analysis of Microsatellite InstabilitiesClinical Diagnostics and Molecular Diagnostics of CancerMicrosatellite instabilities present in 10-15% of colon and gastric carcinomasStudy: 40 cases of colon carcinoma5 microsatellite loci investigated Results compared with traditional PAGE: 95% concordance rate

40Figure 2: Overlay of electropherograms to classify the status ofmicrosatellite loci BAT25 and D2S123.The electropherograms represents the pattern of the separated PCRfragments (MS1 and MS3). No MSI could be determined due to theperfectly matching pattern of both electropherograms (A). Significantdifferences in the pattern of two overlaid electropherograms derivedfrom tumor and non-tumor material of the same patient strongly pointto MSI statusPage 412008Protein ApplicationsProtein PurificationProtein ProductionProteinExpressionFood AnalysisPurity and QA/QC

Page 422008Bioanalyzer Protein Kit portfolioIntroductionP 80

Range 5 - 80 kDaSensitivity:CoomassieSamples:10

SamplesAntibodies (reduced)Small ProteinsP 230

Range 14 - 230 kDaSensitivity:CoomassieSamples:10

SamplesAntibodies (all types)Standard Proteins Range: 10 - 250 kDa Sensitivity: 1 pg/l BSA on Chip Samples #: 10 per Chip Chips #: 10 per Kit Labeling Conc: 1 ng 1 g /l

Coomassie Range (5 ng/L BSA)HSP 250Silver stain Range (200 pg/L BSA)Agilent Protein 80 kitProd Number5067-1515Agilent Protein 230 kitProd Number5067-1517Agilent High Sensitivity Protein 250 kitProd Number5067-1575

42Page 432008Protein 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. 5067-1515Product No. 5067-1517Product No. 5067-1575New*Page 442008Staining, Destaining and DetectionStaining, Destaining and Detection (P-80 and P-230)proteinmicellesSDS + dyedestaindetectionlow background good signal to noise ratio SDS conc. below CMC

If no dilution was done the micelles would result in high background and low sensitivityAvoiding high background, providing better Sensitivity44One additional feature of the protein assays is that the proteins are stained and destained on the chip. This process is explained in a bit more detail on this slide.The staining is done by adding an intercalating fluorescent dye and SDS to the gel matrix in the microchannels. The SDS forms micelles around the protein. It also forms free micelles, without any protein inside, because the SDS concentration is above the critical micelle concentration (CMC) during the separation of the proteins.The fluorescent dye intercalates with the formed SDS/protein micelles, but it also interacts with the free micelles.Without destaining, this would result in a very high background and very low signal to noise ratio, making it basically impossible to detect any protein peaks.During the destaining the SDS concentration is diluted below the CMC, the number of free micelles is decreased because they are less stable, resulting in a low background. The protein/SDS micelles can now be detected with a good signal to noise ratio and a low fluorescent background.Page 452008Clone Selection based on Protein Expression1*2345678*FluorescenceTime (seconds)0102030405060 20 25 30 35 40 45 50 55colony 1colony 2

53.021.529.032.514.46.0kDaprotein of interestExample measured withProtein 50 kit45This next couple of slides show application examples out of such a workflow.The application example shows the use of the Protein 50 for clone selection based on protein expression.4 l total cell culture from different clones were prepared according to the kit protocol and analyzed for the expression of a recombinant 22 kDa protein. Colony 2 shows the expression of the protein in contrast to colony 1, which does not express the protein.The Protein 50 assay provides a fast tool allowing to screen 10 colonies for the desired clone.

Page 462008Monitoring of Protein Purification Process2100 bioanalyzer: gel-like image

Courtesy of P. Sebastian and S.R. SchmidtGPC-Biotech AG, Martinsried, Germanycell lysatewash(fraction 69) elution (fraction 77)flow through(fraction 66)wash (fraction 78)Time (seconds) 15 20 25 30 35 40 45GFP Fusion Protein Analysis2100 bioanalyzer: electropherogramExample measured withProtein 200 plus kit46This slide shows the monitoring of various column fractions during the purification of a GFP fusion protein.

As expected, the desired protein present (and detected) in the starting material, the cell lysate, does not show up in the flow-through and wash. It can be detected in fraction 77 where also the affinity chromatogram showed a distinctive peak (not shown).

Page 472008Expression of a Recombinant Protein in E.coli- Optimization of Fermentation and Induction Conditions

recombinant proteinsoluble protein fractionsolubilized inclusion bodies (50 mM Tris pH 7.5, 100 mM DTT, 8M urea)47Different Expression and Induction conditions were tested for optimal conditionsDifficult to to identify recombinant protein in soluble protein fraction but possible in inclusion bodiesPage 482008Quality control of the Depletion of High Abundance Proteins in Human Serum

Depletion of High Abundance Proteins by Agilent MARS HPLC columnsB

Fractions checked by 2100

A48Fig. 7. A: Gel-like image of serum samples analyzed with the Agilent 2100 Bioanalyzer (Protein 200 Plus Assay). Serum and HSA samples are shown before and after depletion using the Multiple Affinity Removal System on the recommended Agilent 1100 series HPLC. Lanes: (1) Serum prior to chromatography, (2) depleted serum fraction, (3) high-abundant protein fraction, (4) HSA solution prior to chromatography, (5) HSA fraction after chromatography, (6) depleted fraction of HSA sample. B: Electropherogram comparing high-abundant protein fraction (blue) and depleted serum fraction (red).

Page 492008

Quality Control of Antibodies

90 kDa160 kDaintact antibody16% half antibodyDetermine the half antibody content in IgG preparationsAntibody analysis under reducing and non-reducing conditions

Ab reducedAb non-reducedlight chainheavy chainintact antibodylight + heavy chainAb reducedAb non-reducedAbsolute Quantitation of IgG samples

49Page 502008Antibody 1 - StandardLadderAntibody 1 1 month 40CAntibody 2 12 weeks 40CA

Antibody 2 - Standard97.421066.732.553.0117.029.021.514.4LMHCLCBFluorescenceTime (seconds)0100200300400500600 21 23 25 27 29 31 33 35Antibody 1- StandardAntibody 1- 1 month 40CFluorescenceTime (seconds)0100200300400500600 21 23 25 27 29 31 33 35Antibody 2- StandardAntibody 2- 12 weeks 40CAnalysis of Antibody Stability stress testPage 512008Combination of IEF with SDS-PAGEAgilent 3100 OFFGel Fractionator + 2100 bioanalyzer

Page 522008Description 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 rangeTarget Applications:Protein QA/QCreliable quantitation of main compound besides minor impuritiesProtein 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)Page 532008Extended experimental workflowTransfer 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 2100labeling kitseparation kit5-90 min40 min5 min35 minSampleData53Page 542008Principle of High Sensitivity 250 Protein StainingpH 8.5 0C

sample proteinchemically activated fluorescent dyechemically activated Covalently labeled 30 minNHSNH2EtOH10 minEthanolamine

Step 1Labeling Reaction

SDS + 95C5 minStep 2SDS-Denaturationlabeled ProteinSeparation&DetectionLaser inducedFluorescence30 minStep 3Separation on ChipPage 552008Reproducibility of Labeling Reaction: Ladder

Rugged Labeling reaction:Reproducible reaction provides comparable signal intensities. Homogenous labeling without extra bandbroadeningNo deviation in peak width is indicating a constant number of dye per protein molecule and proves a stable protocol55Page 562008Sensitivity: Silver Staining vs. Bioanalyzer

Highest sensitivity:Labeled proteins can be measured down to pg/L concentrations loaded on ChipDirect 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)56Page 572008Linear Dynamic Range Test: IgG

Linear dynamic range:Quantification of labeled IgG from 10 pg/L to 100 ng/Laverages 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 run57Page 5820082100 Bioanalyzer Compliance2100 expert software- One version for all assays Declaration of system validation2100 expert security pack 21 CFR part 11compliance Electronic records- Electronic signatures- Audit trails2100 bioanalyzer - IQ and OQ/PV services Declaration of conformityChips and reagents- Declaration of conformity

58This slides give and overview of the current situation and our plans for the future for the bioanalyzer.Currently we have 2 software versions, Biosizing for RNA, DNA, and protein assays and Cell fluorescence for the cellular assays.We have 3 main reasons why we decided to consolidated them:It is more convenient to have one software for al assays, with no need to switch between 2 SW applicationsDifficult to add new software features and new assays to the two old SW packagesPrevious SW was not developed under compliance regulationsSo the new software is a prerequisite for compliance. With the new software we will also provide the documentation for compliance such a Declaration of system validation, and Declarations of conformities for the HW and the consumables, which is again required for compliance. Later in the second phase we will offer a Security Pack add on software with all the features required for full 21 CFR part 11 compliance. The complete compliance topic will be discussed in more details in a separate E-seminar.

Page 592008Cell ApplicationsProtein ExpressionTransfection MonitoringApoptosis DetectionGene SilencingCell StainingInside / outside

Video

Page 602008Cell Assay ApoptosisTransfection Efficiency MonitoringDetection of GFP-transfected cellsAntibody 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

60This is a list ofsome target applications which we have described in specific application note.The AN give recommendations for reagent suppliers and detailed protocolls as well as guidlines for the data evaluations.For apoptosis we have the detection of PS by annexin V or the detection of active Caspase 3.Next area is monitoring transfection efficiencyfor e.g. optimizing protocolls for new cell lines of new transfection techniques.You can do this by detection of green fluorescent protein or by AB staining.Monitoring protein expression in cell lines is also an interesting area. Here intracellular or cell surface Antibody staining can be applied.

Further application areas foreseeable are Cell Cycle analysis, Proliferation assays, Live/dead assays, TUNEL assays and more...Page 612008

Principle of Pressure-Driven FlowFor cell assays (analysis of cell fluorescence parameters)On-chip simple flow cytometric studiesPressure driven flow is used to move cells in a controlled manner through the micro-channelsCells are hydrodynamically focused to a portion of the channel by a side stream of bufferCells pass the fluo-rescence detector in single file and each event is monitored in a histogram or dot plot

The micro-channels of the glass chip are filled with cell buffer61Some areas of the chip used for cell assays are zoomed out to demonstrate what happens during the run. The depicted scenario shows the hydrodynamic focusing of a sample from sample well 3.Event = data point generated after the detection of a red, blue, or red/blue fluorescing cellRead!Page 622008The Bioanalyzer Lab-on-a-Chip Approach

Separation on disposable, -fabricated glass chips made of two glass layers: one with micro-channels (x10m, 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 wellschip preparation in less than 5 minutes

Benefits convenient handling minimized risk of cross-contamination versatile design for multiple experiments on one platform

62Read!Chip priming (with gel or buffer) and sample loading onto the chip is the only manual interaction which is needed prior to the run. After the chip is placed in the bioanalyzer everything runs automaticallly - sample handling, separation, detection and analysis.Page 632008Flow Cytometry on a Chip - Hydrodynamic FocusingAll six cell samples are hydrodynamicly focused to one side of the micro channelAt each of the six pinch points the cell stream is joined by a buffer stream from one of the two buffer wellsThe two liquids do not mix immediatelyThe cells then move towards the detector in single file

63Here we see the process of hydrodynamic focusing on the chip again in more detail....for all six samplesInterestingly, the two liquids do not mix immediately and the detection area is located directly after this pinching area.

Page 642008Flow Cytometry on a Chip - Two Color Detection- Three Types of Events

Blue/red cellsRed cells

Blue cellsDot plot view for easydata evaluation64Page 652008Some Target applicationsApoptosis:Annexin VDetection of phosphatidylserine on the cell surfaceCaspase-3Detection of activated caspase-3 in the cytoplasmTransfection 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 nucleusGene silencing:Optimization of siRNA transfection procedureVerify silencing by cellular protein expression measurementCorrelation of siRNA uptake and gene knockdown

65This is a list ofsome target applications which we have described in specific application note.The AN give recommendations for reagent suppliers and detailed protocolls as well as guidlines for the data evaluations.For apoptosis we have the detection of PS by annexin V or the detection of active Caspase 3.Next area is monitoring transfection efficiencyfor e.g. optimizing protocolls for new cell lines of new transfection techniques.You can do this by detection of green fluorescent protein or by AB staining.Monitoring protein expression in cell lines is also an interesting area. Here intracellular or cell surface Antibody staining can be applied.

Further application areas foreseeable are Cell Cycle analysis, Proliferation assays, Live/dead assays, TUNEL assays and more...Page 662008Typical workflow: Cell assays: sample preparationadherentsuspension(trypsinize) & harvest by centrifugation, wash wash twiceResuspend cells in cell buffer (LabChip Kit)Load on chipAdd staining reagentand incubateData analysis to resultCustomerAgilent

66The procedure to prepare fluorescent labeled cells is the same as in conventional flow cytometry.-culture cells-harvest cells and wash them to get rid of the medium.-incubate cells with dyes/antibodies for 15-30 min at RT or 4 C-wash again

For final chip loading cell samples are resuspendet in a special buffer optimizing for density and viscosity. Thats only one additional step.

Insert chip and start run...Page 6720082100 BioanalyzerRed 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

Standard Flow Cytometers have 3-4 fluorescence detection channels:FL1: excitation 488 nm, detection 530 nmFL2: excitation 488 nm, detection 585 nmFL3: excitation 488 nm, detection 661 nmFL4: excitation 635 nm, detection 670 nm67The bioanalyzer has two light sources for fluorescence detection.One red laser with...And one high power LED for exitation in the blue at...This anables the bioanalyzer to work with a broad range of fluorescent dyes.Page 682008Cell Assays - Applications: ApoptosisAnnexin BindingPhosphatidyl-serine from inner leaflet flips to outer membrane during apoptosis and can be labeled by Annexin VHealthy cellLive apoptotic cellDead cellLive dye: Calceinbiotin-Annexin+ Cy5-streptavidin68In the earlyphase of Apoptosis a shift of a special membran lipid, the phasphatidyl-serin, from the inner leavlet to the outer leavlet of the cell membran can be observed.

Those apoptotic cells can be stained by AnnexinV which binds very specific to this membran lipid. The AnnexinV is labeled by a red dye.With a additional blue dye for live cell staining helps to destinguish between healthy, apoptotic and dead cells.Page 692008Annexin V Assay (24h Induction)0%20%40%60%80%0102030Sample Number% Apoptotic2100-12100-22100-3Flow cytometer5 chips, each loaded with control in well 1 and 24h sample in wells 2-6Three Bioanalyzer instruments vs a flow cytometer reference instrumentChip 1Chip 2Chip 3Chip 4Chip 569This graph highlights the good reproducibility of the results.Five chips on 3 bioanalyzer instruments were run.And the results from chip to chip and from Instrument to instrument are very reproducible.Results match also reference measurements with a conventional flow cytometer instrument.Page 702008

Applications: Protein Expression AnalysisGFP Transfection Efficiency ControlCHO-K1 cells were transfected with EGFP DNA and Lipofectamine.

GFP transfected cellsMock transfected cells

0.1 %56.6 %ControlEGFP transfected70In this experiment chinese hamster ovary cells were transfected with a DNA plasmid which encodes for GFP.

Upper panel shows the control transfection, cells don't express GFP.Examples for data evaluation in dotplot view and histogram view are shown in comparison to the microscopy view.

The transfection efficiency with 56% can be easily determined with the bioanalyzer whereas. Data is based on all live cells. Getting such a solid, reproducible result from the microscope might be very difficult.

-----exp. data.------------Mock- (I) and GFP-transfected (II) cells were harvested and assayed on the microfluidic system. Shown are dot plots (A), histogram views (B) and photographs of the cells taken using a fluorescent microscope (C).

Lipofectamine 2000 from (Life Technologies) Page 712008GFP Transfection Efficiency0%10%20%30%40%50%60%70%12345Chip NumberTransfection EfficiencyFlow cyt.2100-12100-22100-32100-12100-22100-3AllFlow cyt.ctrlmean0.460.310.470.400.16SD0.080.290.430.290.12GFPmean60.1959.1559.2659.5360.90SD2.132.482.102.261.22%CV3.544.193.543.802.0171The data here again demonstrates a very good reproducibility of the bioanalyzer results.Very low SD and CV values--------might skip this slide--------each of the 15 chips was loaded with one control cell sample and 5 samples of GFP transfected cellsPage 722008Flow Cytometry Assays Applications - Cell surface Antibody stainingCell expressingprotein of interestTarget proteinLive dye: CalceinCy5 or APC-labeled AntibodyCell not expressingprotein of interest72This schematic shows the principles for the Cell surface AB staining. There is a broad range of application for this assay. Many different primary or secondary antibodys are available for different targets in cell line characterization or protein expression studies.Again the blue live dye Calcein is applied as reference dye.The target of interest is detected by a specific AB which is labeled in the red by Cy5 or APC for instance.Page 732008Extracellular Antibody StainingAveraged data per instrument0.0010.0020.0030.0040.0050.0060.0070.0080.00123456sample #% gated% gated 2100-1% gated 2100-2% gated 2100-3%gated 2100-4Flow cytometerJurkat cells were stained with calcein alone or with calcein and APC-labeled anti-CD3 antibody. Mixtures of both populations were prepared at various ratios.Samples were analyzed with four 2100 instruments on 5 chips and compared to a flow cytometer reference instrumentMean % CD3+ cells2100-12100-22100-32100-4Flow cyt.60.967.866.665.060.934.436.736.734.329.817.317.618.717.213.88.99.49.98.36.55.14.45.34.93.20.80.60.30.30.073In this experiment Jurkat cells, T-cell leukemia, where stained with Antibodys against CD3. CD3 is a surface marker of these cells.Dilutions with non-stained jurkat cells (sample 6) were made to mimic different percentages of CD3 positive cells. The 6 samples were analyzed on a chip with 4 bioanalyzer instruments and a FACS reference instrument.Interestingly, the dilution sample with the very low amount of positive cells (near to 5 percentage ) could be detected with the 2100 bioanalyzer.Data is in good comparison to the standard FACS instrument.Page 74200815 mincells + CBNF

15 minOn-chipSpin, aspirate,resuspend,spin, aspirateCBNF

10 min 10 min 10 min15 min 35 minConventional

Resuspend cells in CBGFP On-Chip Staining - Workflow

74Comparison of workflows. Upper is for conventional staining. Lower for new faster & easier staining procedure described in AN.- Use less hands-on time- less tubes and pipetting steps- save 20 minutes for each sample- save reasonable amount of expensive staining reagentsPage 752008

GFP On-Chip Staining - Histogram Quality2100 bioanalyzerFlow Cytometer

75Page 762008Agilent Web pages with 2100 content

www.opengenomics.com

www.agilent.com/chem/labonachip

76This slides give and overview of the current situation and our plans for the future for the bioanalyzer.Currently we have 2 software versions, Biosizing for RNA, DNA, and protein assays and Cell fluorescence for the cellular assays.We have 3 main reasons why we decided to consolidated them:It is more convenient to have one software for al assays, with no need to switch between 2 SW applicationsDifficult to add new software features and new assays to the two old SW packagesPrevious SW was not developed under compliance regulationsSo the new software is a prerequisite for compliance. With the new software we will also provide the documentation for compliance such a Declaration of system validation, and Declarations of conformities for the HW and the consumables, which is again required for compliance. Later in the second phase we will offer a Security Pack add on software with all the features required for full 21 CFR part 11 compliance. The complete compliance topic will be discussed in more details in a separate E-seminar.