Eco Real-Time PCR System User Guide
Contents
1. 8 Load the Plate 9 Define a New Erkperiment 10 SetUpthePlateLayout 12 Set Up the Thermal Profile 18 MONRO RUM ox ux sls usss s S naam au ag s ss hee eee ee eee E 19 AnalyzeData 20 Chapter 3 System Information 27 COMDONCHIS iris teen hee ne Gh eee eee ees beeen E 28 Specifications and Environmental Requirements 30 ees ates eel da aoe aa ee ee ica 31 Electromagnetic Compatibility 32 Cleaning And Maintenance 33 Return zio REST T EET ede eee COT T TT IRA 34 Chapter 4 CONCEpts o 35 Chapter 5 Glossary 37 Eco Real Time PCR System User Guide Table of Contents Part 1501 15 Rev B Overview sede cs 44 26 ohne oe Gun DA oe eeenune ale eine 2 Real Time POR as agen ua RELA Ce ales 3 Absolute and Relative Guantification 4 Allelic Discrimination and High Resolution Melt 5 Multiplexing Real Time PCR 6 Mur Oa s te se bii net AATGCOGC rea pegag rct TGA c T E Eco Real Time PCR System User Guide 1 gff Je3deuo Ov2. concentration Part 15017157 Rev B Standard A serial dilution of a target of known concentration used as template to generate a standard curve Standard Curve A plot of Cq values against the log of target amount Used to determine an assay s dynamic range efficiency slope RZ and sensitivity y intercept Standard Deviation SD The SD of replicate Cq measurements is a measure of the precision of the assay TaqMan Probe See Hydrolysis Probe TAMRA Tetramethyl 6 carboxyrhodamine Commonly used as a quencher Target The DNA or RNA sequence to be amplified Template See Target Template can also refer to a saved experiment that can be used as a model for new experiments in the software Threshold A level set above the background signal generated during the early cycles of qPCR When adjusted manually it should be set in the middle of the exponential stage of qPCR TET Carboxy 2 4 7 7 tetrachlorofluorescein Tm The temperature at which 50 of dsDNA is single stranded melted Unknown A sample containing an unknown amount of template Y Intercept In a standard curve the value that crosses the y axis at x 1 single copy target Eco Real Time PCR System User Guide 4 Glossary 42 Part 15017157 Rev B Technical Assistance For technical assistance go to http www illumina com ecoqpcr MSDS Material safety data sheets MSDSs are available on the Illumina website at
3. http www illumina com msds Product Documentation If you require additional product documentation you can obtain PDFs from the Illumina website Go to http www illumina com support documentation ilmn When you click on a link you will be asked to log in to iCom After you log in you can view or save the PDF To register for an iCom account please visit https icom illumina com Account Register Eco Real Time PCR System User Guide AS SOUEISISSY7 eoluyos ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCT ACTT TTAACCTTAA CT C C CGTACCATT C TTAACCTTAAGA TGATAACAGTAACAC CTG ACTTGTTGATCCACTGATTCAACGTACCGTATCAATTGAGACTAAATATTAACGTAC AAGAGCTACCGTCTTCTG GATTACTTGATCCACTGAT TCAACGTAC TCCACTGAT TCAACGTACCAAGAT TACTTGATCCACT GAT TCAACGTA CGAA TCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTC C TTACTTGATCCACTGATTCAACGTACCGTAA CAGTAACACAC ACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGT AACGAA TCAATTGAGACTAAATATTAA TTAAG AATGATAACAGTAACAC CTGTTAA GATTACTTIGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATT CCATTAAGAGCTACCGTCTTC C ACTTGATCCACTGATTCA AA A CTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT T ACTIGATCCACTGATTCAACGTACC CGTATCAATTGAGACTAAATATTAACGTACTTAACC i TGATTCAACGTA TAACGAACGTCTTCTG GATTACTTGATCCACTGATTCAACGTACCGTAACGAA TCAATT CTAACGA GAGACTAAATA ACCATTAAGAGCTACAACC TTGAT TGATTCAACG GT
4. 5 0 5 1 1 5 2 High Resolution Melt HRM enables the Variant 2 detection of almost any genetic variation SNPs mutations and methylation patterns Because HRM assays only require primers and a dye no probes or DNA sequencing the method is simpler and cheaper than traditional approaches After the amplification phase the amplicon is slowly heated until it melts The melting temperature and profile are directly linked to the amplicon sequence Figure 6 HRM Difference Plot HRM s power comes from the resolution of the sample s melt profile It requires a high quality Wild types Optical system and precise thermal uniformity HRM PCR amplicons below 300 bp provide the best resolution The shape of the resulting melting curves which is sensitive to almost any genetic change determines sample identity To easily cluster equivalent samples a reference curve e g mutant is subtracted from the other curves to generate a difference plot Figure 6 Normalized minus mutant Heterozygotes 80 81 82 83 84 85 86 Temperature C Resources Livak KJ 1999 Allelic discrimination using fluorogenic probes and the 5 nuclease assay Genet Anal Biomol Eng 14 143 149 POLAND server http www biophys uni duesseldorf de local POLAND poland html Wojdacz TK Dobrovic A Hansen LL 2008 Methylation sensitive high resolution melting Nature Protocols 3 12 1903 1908 Eco Real Time PCR System User Guide 5 uonnios ti YOH p
5. D HA Birdesg Dra CHAS tandad Curse T fe 1 UK ty fgg Md Dem Cy Deaiby Ang up Motu Bun Da di Fco GA L s Eco Real Time PCR System User Guide 2 5 Results Tab Eco software automatically analyzes the data and generates a plot based upon the experiment type along with any baseline or threshold adjustments gt Absolute quantification experiments generate a standard curve The slope PCR efficiency and R appear in a table in the upper left Workflow Relative quantification experiments generate a bar graph that includes error bars v Allelic discrimination experiments generate a scatter plot gt HRM experiments generate a melting curve graph which can be displayed as either a normalized melting profile or a difference plot to maximize cluster resolution Figure 22 Results Tab Example of a Standard Curve display He compatte Dala Ex clu de weli C2 In some cases you might want to exclude outlier data from the analysis Right click the data point in the graph or the well location in the table and select Exclude Well from the context menu see inset The Well Table will list the well as Excluded 2 6 Part 15017157 Rev B System Information ComponentiS ok koe ac ok Oh Pie i isi nis Ea n S Red dcs 28 Specifications and Environmental Requirements 30 e dee eras d
6. For example High Resolution Melt HRM is associated with DNA Binding dyes If this will be a quantification experiment select Comparative Quantification or Standard Curve Enter an experiment name of up to 20 characters Optional If you want to save the experiment as a template select File gt Save As and save in the ecot file format Click 4 The Setup window opens with the Plate Layout tab visible Part 15017157 Rev B Load a Template Optional After designing an experiment you can save the experiment as a template using the File Save As menu option The template retains the experiment setup plate layout and thermal protile 1 Startanew session in the Eco software and click the Templates tab Recent and saved templates appear Recent Templates E program Fles eias PD V oke Templates valuabonelate Template pot Q D O sa Dedi KY s To 0 0 0 0 a E RR BE e o Re ed A TT RR GG UU o o SY Set Up Samples ES DONNE rut mne o 0 Unknown 0 Cuebed dl A A w w e o NTC e UV UV wow vw v Rardad vw wv wv wv 3 If you wish you can change the assay and sample designations on the plate or adjust the thermal profile You cannot redefine the application detection chemistry starting material or quantification method Eco Real Time PCR System User Guide 1 1 jJuauulledx3 MAN e euuyer Set Up the Plate Layout Figure 10 Plate Layout Tab
7. Option Help Quantification DNA Binding Dye DNA Standard Curve Setup Plate Layout Thermal Profile 2 3 5 6 7 8 O Set Up Assays Assign Name Assay Role File Edit Workflow Assay 1 Unknown E O mm sirene OJO Y Set Up Samples Assign Name Sample 1 Sample 2 C Samnle 3 J ROX Normalization New Session 1 KI The Plate Layout tab lets you assign assays and samples to Figure11 Image of Eco wells An image of an actual plate layout appears at the right Evaluation Plate Plate layout involves four steps 1 Setup assays page 13 2 Set up samples page 14 3 Assign assays and samples to wells page 15 4 Define standards page 16 You can lay out the plate any time between detining the experiment and analyzing the data The Analyze Data window page 20 will not become active at the end of the run until the plate layout is complete 2 Part 1501 15 Rev B Set Up Assays An assay is the set of primers or primers probe used to quantify a gene Assays can have different roles such as Unknown Standard Negative Positive or NTC Non Template Controls Each assay is associated with a reporter dye which identifies the assay during analysis Reporter dyes can belong to one of four channels each of which is defined by a specific excitation and emission range You can assign up to four assays per well Within each well assays cannot use reporter dyes from the same chan
8. add a new stage such as a reverse transcription incubation at the beginning The stage will appear at the end of the cvcle and you can drag it to the desired location Alternatively you can drag the 45 icon to the location within the profile where you would like the new stage to be added The camera icon 4 indicates when the Eco collects image data In three step PCR you can select whether to collect data at the annealing or extension step Extension is the default To move it to annealing mouse over the annealing step and click the dim camera icon that appears and then drag it to the annealing step gt To remove a stage drag it to the T trash can or highlight it and press Delete 1 e Part 15017157 Rev B Monitor Run iy Crede 3 Plate Layout View Shows sample type Amplification Plot Highlight wells to display a subset of data Eco Real Time PCR System User Guide Channel Select channels to view in Amplification Plot Thermal Profile Shows current stage highlighted in orange Amplification View Shows amplification as it occurs in each well Amplification Plot Shows PCR signal in real time 4 5 6 7 8 YA Wa NA Na Na A E de 19 UNH JOHUOIN Analyze Data Workflow There are four tabs gt Component Data gt Amplification Plot gt Melt Curve when applicable Results The window controls are the same for each tab 20 Zoom In Drag t
9. curves to calculate the initial threshold for determining Cos The region of the amplification curve that corresponds to exponential growth changes depending on whether you select linear or log view To set the threshold automatically 1 Click Y Analysis Settings in the right panel The ee nc Analysis Settings dialog box opens Analysis Settings 2 Select the Auto Threshold check box then click Apply Assay To adjust threshold numerically sa 1 Click si Analysis Settings in the right panel The Po Analysis Settings dialog box opens E 2 Enter the desired Threshold value then click Apply cd w EJ Apply To adjust threshold graphically Drag the horizontal bar up or down into the exponential growth phase of the curve gt In a linear scale view this needs to be set close to the inflexion point of the amplification plots tu P In a log scale view it should be set in the middle of the exponential phase Part 15017157 Rev B Melt Curve Tab This tab is active if you ran a melting curve in your thermal profile while using DNA binding dyes such as SYBR green The Melt Curve tab displays the negative derivative of RFU versus temperature dRFU dT and calculates melting temperatures Tm based upon peak calls Tm calls up to three per well are listed in the Well Table on the right side of the tab and are ranked based upon maximum amplitude ejea ezApuv Figure 21 Melt Curve Tab Duiribcaion
10. front Ready Status and Error The following table shows the meaning of each combination of off on and flashing lights NOTE Flashing lights are indicated in the table by dashed lines around the outside of the light Sjueuoduuo Lights Description Lights Description READY Power Off READY Non Fatal Error C CL STATUS STATUS Decide whether you want DOLO NG O O O O O to terminate the run ERROR p aaa ERROR Initializing READY Fatal Error Run status conducting self tests and STATUS Terminated O O O O O heatingthe thermal bonnet S O O O O Instrument might have EROR C ERROR overheated or encountered C CHEN A hardware failure READY Ready Idle Software Updating SS STATUS STATUS Chipa p ra 0 01 ERROR C READY Run In Progress Communicating with A STATUS Do not switch off or open Netbook 0 0 0 the door while a run is in ERROR progress C READY Run Complete CZ STATUS 00000 ERROR C 2 Eco Real Time PCR System User Guide 2 9 System Information opecifications and Environmental Requirements Optical Thermal Operational Physical Environmental 30 Light Source Detector Thermal Cycling Thermal Uniformity Sample Format Reaction Volume Warmup Time Typical PCR Run Time Sensitivity of Detection High Resolution Melt Multiplexing Passive Reference Dimensions Weight Electrical Temperature Range Humid
11. is inversely correlated to the logarithm of the initial copy number Dark Quencher quencher without any native fluorescence Black Hole Quencher BHO dyes are an example Delta Rn ARn The normalized Fluorescence of an amplification plot with background and ROX normalization dye correction Derivate Melt Curve plot of temperature x axis versus the derivate of fluorescence with respect to temperature dF dT y axis Used to analyze the Tm of an amplicon DNA Binding Dye A dye that increases its fluorescence in the presence of double stranded DNA dsDNA Double stranded DNA Dual Labeled Hydrolysis Probe See hydrolysis probe Dynamic Range The range of template concentration over which accurate Cq values can be determined Extrapolation is not recommended Efficiency See Slope Endogenous Control An RNA or DNA template that is naturally present in each sample End Point Analysis Qualitative analysis of PCR data at the end of PCR Allelic discrimination assays genotyping are an example Exogenous Control A RNA or DNA template that is spiked into each sample at a known concentration Part 15017157 Rev B FAM 6 carboxy fluorescein The most commonly used reporter dye at the 5 end of a Taqman probe Filter Components used to limit the bandwidth or the excitation or emission energy to the next component of the optical path Fluorophore The functional group of a molec
12. starting quantity target 90 and 11096 typically indicates a problem with the template or assay design The R value a measure of assay performance should be 0 99 for the assay to accurately quantify unknown samples Figure 4 Relative Quantification Experiment HI a Samp shi i m r The relative quantification method measures the level of gene expression in a sample relative to level of expression of the same gene in a reference sample In addition the level of expression of every gene in the assay is normalized to the expression of a reference gene As a best practice use multiple reference genes when quantifying gene expression The results obtained are expressed as relative levels in gene expression compared to the reference sample R Figure 4 Part 15017157 Rev B Allelic Discrimination and High Hesolution Melt Allelic discrimination assays using hydrolysis Figure5 Allelic Discrimination Scatter Plot probes provide a rapid and sensitive method to genotype samples Two variants alleles are Par interrogated at the same time multiplex qPCR oo olo One probe is typically labeled with a FAM dye e and the other with a VIC dye Samples with Qv Variant 1 2 FAM signal and no VIC signal are homozygous E for variant 1 samples with VIC signal and no FAM signal are homozygous for variant 2 and samples with both FAM and VIC signal are 2o bo 0 5 amp NTC ee heterozygous Figure
13. 20 CAUTION Do not open the lid while a run is in progress This allows extraneous light into the system and can corrupt the data 7 When the run is complete open the Eco lid Press the plate release lever and remove the plate from the block Dispose of any hazardous materials in biohazard caustic material or other appropriate containers according to your local safety regulations e Part 15017157 Rev B Load the Plate 1 Thaw all necessary reagents templates primers probes and master mix 2 Turn on Eco and the netbook PC and wait until the Eco Ready light is solid blue 3 Confirm that the block and optical path are clear of visible contaminants and there is no physical damage to the system such as dents frayed cords or damaged levers 4 Place a 48 well plate into the Eco sample loading dock aligning the notch with the matching indentation on the adapter 5 Turn on the dock light and incline the dock to a comfortable angle for pipetting 6 Pipette samples and qPCR reagents into the plate according to your protocol WARNING Wear protective gloves and eyewear when handling any material that might be considered caustic or hazardous 7 Seal the plate with an Eco optical seal Holding the plate in place on the Eco sample loading dock drag the squeegee firmly across the surface to ensure the seal is secure 8 Place the plate adapter with your loaded and sealed plate in the centrifuge carrier along with the second suppl
14. AACGAACGTATCAATTGAGACTAAATATTAA ATTAAGAGCTACCGTGC j TAAATAT TAACGT TTAAGAT TAC CCAC TACC AT GACT TTAACGTACC CGAAAAGAATGATAACAGTAAC AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCA ZGTACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGACGAACTTCTGTTAACCTTAAGATTACTT GGAGCTACCGTGCAACGAAAATAACCTTAAGATTACTTGATCCACT DL lc a CGAAAAGAAT ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTT A ACCGTAACGAACGTATCAATTGAGAC Muse sa CGTACCATTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTC vice ATTAAGAGCTACCGTGCAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACT Li TAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GA AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCA Rara TTACTTGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTICAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAGCAACG AAAA AACAGTAACACACTTCTGT TAACCTTAAGAT TACT TGATGCACTGATT CAACGTACCGTAAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCT TTCTGTTAACCTTAAGAT TA CGTACC TA TTGAGACTAAA CGTACCATT
15. ACTT NGAGCTACCGTGCAACGAAAATAACCTTAAGATTACTTGATCCACTGATTCAACGTACTTCTGT TAACCTTAAGATTACTTGATCG iIACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTG AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTICAACGTACCGTAACGAACGTATCAATTGAGA TAACACACTIC GAAAAGAATGA 3GTACCATTAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAG AAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGA CCACTGATTCA o TCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC TA CTTCTGT TAAC TTACTTGATCCACTGATTCAACGT TAAGAT TACT TGATCCACT GATT CAACGTACCGTAACGAACGTAT CAATTGAGCTTCTGTTA ACTAGCAACG 1ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTG DCATTAAGAGCT illumina FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Catalog EC 900 1001 Part 15017157 Rev B Current as of August 2010 INTENDED USE The Eco Real Time PCR System is intended to support the Real Time polymerase chain reaction PCR application needs of life science researchers This includes gene expression quantification and analysis as well as genotyping by allelic discrimination or high resolution melting The system is able to support other applications and protocols as well Eco features high quality optical and thermal modules to provide optimal performance and data quality The system includes data analysis software that is preloaded on a netbook computer a
16. AGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAA ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTI GATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTAGGAT TAAGAGGTACCGTCTICTGTTAACCTTAAGATTACTIGATCCACTGAT T CA ZAATTGAGACTAAATATTAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACC CGTATCAATTGAGACTAAATATTAACGTACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAACGA NE TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGT GCAACGACGAAAAGAAT GATAACAGTAAC AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTTCT vcr E HE n O AR ZGTACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAACGACGAACTTCTGTTAACCTTAAGATTACTT GGAGCTACCGTGCAACGAAAATAACCTTAAGATTACTTGATCCACTGATTCAACGTACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAGCTACCGTGCAACGACGAAAAGAAT ACGAAAAGAAT AACCTTAAGATTACTT AC CGT CGT IT ACTAAATAT T GA E ACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTA AACGAA CAATTGAG TAT TAAC
17. AT GA AATGATAACAGTAACACACTTICTGTTAACCT TAAGAT TACT TGATCCACTGAT T CAACGTACCGTAACGAACGTAT CAAT T GAGACTAAATAT TAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCA AACGTACCGTAACGAACGTATCATTAAGATTACTT GATCCACT GATT CAACGTACCGTAACGAACGTAT CAAT TT GAGACTAAATAT TAACGTACCATTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGT TAAC TTACTTGATCCACTGATTCAACGT TAAGAT TACT TGATCCACT GAT TCAACGTACCGTAACGAACGTAT CAAT TGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACTGAT IT CAACGTACCGTAACGAACGTAT CAAT TGAGACTAGCAACGA iIACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCA PESE e Ai ACCGIAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCT e nu cci iIACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT SGTACCAT TAAGAGCTACCGTGCAACAGTAACACACTTCTGTIAACCTIAAGA TTACTTGATCCACTGATICAACGTACCGTAACGAACG TATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGA AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCA TTACTTGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAA
18. CGTACCGTAACGAACGTATCAATTGAGACTAGCAACQG iTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGTCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGAAAAGAAT GATAACA earl ACCGTAACGAACGTATCAT TAAGATTACTT GATCCACT GATT CAACGTACCGTAACGAACGTAT CAAT T GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGT TAAC CT CAACGTTAAGATT GTA CGAACGTATCAA CTT TTAACCTTAAGATTACTTG T C C C T AAG G CGTATC G C GAGC TGTTAACCTTAAGA CT AACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCT ART OATAACAGTAACACACT FCTGTTAACCTPAAGATTACTEGATCCACTOATTCAACGTACCOTAACOAACGTATOART EGAGAC TAAATALPAACO TACCATTAAQAQETACOGTOTECTGTTAACCTTAAGATTACT TGATCCACYGATTCA SAATTGAGACTAAATATTAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACC CGTATCAATTGAGACTAAATATTAACGTACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAACGAI TGAGACTAAATAT TAACGTACCATTAAGAGCTACAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAACAGTAAC AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG A AS AGO OCS RS SAGA LIA GU GSICCACTSATTCA 3GTACCATTAAGAGCTACCGTGCAACTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGA ACCTTAAGATT
19. GCT TGTTAA AAGATI GATAACAGTAACACAC CTTGATCCACTGATTCAACG GTAACGAACGTATCAATT GA TAT TAA CATIAAGAGCTACCGTCTTCTGTTAACCTT ACTTGATCCACTGATTCA AACGTACCGTAACGAACGTATCATTAAGATTACTTGATCCACTGATTCAAC GIAACGAA CAATTGA TAT TAACGTACCAT TAAGAGC GTGC TAACAGTAACA T CA CACAC ACTTGATCCACTGATTCAACG GATTACTTGATCCACT GATT CAACGTACCGTAACGAACGTATCAA G AACGTACCATTAAGAGCT 3GTACCATTAAGAGCTACCGTGCAACAGTAACACACTTCT CTTGATCCACTGATTCAACGTACCGTAACGAACGTAT A TATTAACGTACCATTAAGAGCTACCGTGC AG ACA ACTTGATCCACTGATTCAAC GTAACGAACGTATCAATTGA TATTAACGTACCATTAAGAGCTACCGTCTTC ACTTGATCCACTGATTCA TTACTTGATCCACTGATT CG ACTTGATCCACTGATTCAAC CGTAACGAACGTATCAATTGAGCTTC CTTAA ACTTGATCCACTGATTCAACG AACGAACGTATCAATTGAGACTAGCAACG TATC G TATTAACGTACCATTAAGA ACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTG AAATA CCATTAAGAGCTACCGT A AACGTA AACGAACGTATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GC Sey CTICT TACCGT C C C C AACGACG GAATGA AACACACTICTGTTAAC TTACTTGATCCACTGATTCAACGTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAGCAACG ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGTTGATCCACTGATTCAACGTACCGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTAC iii GATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTG
20. GTACCAT TAAGAGCT ATAACA CACA IT TACT TGATCCACTGATTCAA GTAACG CAATTGA TA CCATTAAGAGCTACCGT GC AACAGTAACACACTTO SETACCATTANGAGCIACEGICCAACAGTAACACAGITOT CTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAG TATTAAC ATTAAGAGCTACCGT GC ATAACAGTAACACA TTACTTGATCCACTGATTCAAC GTAACG GTATCAATTGA TATTAACGTACCATTAAGAGCTACCGTCTIC ME Spe E AR AATGATAA AA T AACCTTAAGA GTACC CGTAT AAA G AACCTTAAGAT T ACGTACCGTAACGAACGTATCATTAAGAT TACT T GATCCACT GATT CAACGTACCGTAACGAACGTAT CAATT GAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAA TTACTTGATCCACTGAT TCAACGT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT TGAGCTTCTGTTAACCTTAAGAT TACT T GAT CCACTGAT TCAACGTACCGTAACGAACGT A CART GAGACT GCAACGH ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCATTAAGAGCT lumina Inc 9885 Towne Centre Drive San Diego CA 92121 1975 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
21. IEC 61326 1 2005 and IEC 61326 2 6 2005 To confirm proper operation gt The electromagnetic environment should be evaluated prior to operation of the system Donotuse this system in close proximity to sources of strong electromagnetic radiation e g unshielded intentional RF sources as these may interfere with proper operation If you notice any interference discontinue using the system until all issues are resolved Resolution may include moving cords from other equipment away from the system plugging the system into an outlet on a different circuit from other equipment or moving the system away from the other equipment If you continue to have difficulties contact Illumina 32 Part 15017157 Rev B Cleaning And Maintenance Clean the block and housing as needed following these directions CAUTION If hazardous or biohazardous material is spilled onto or into the equipment clean it immediately 1 Turn the system off and allow the block to cool completely 2 Using a lint free cloth slightly dampened with clean water gently wipe the surfaces of the equipment If a stronger cleaning agent is needed use a lint free cloth slightly dampened with 95 isopropyl alcohol Follow these practices for proper maintenance of your Eco system gt Every time you use the system visually check it to confirm there is no obvious physical damage such as dents frayed cords or damaged levers If you see any damage discontinue use and con
22. TRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY O 2010 Illumina Inc All rights reserved Illumina illuminaDx Solexa Making Sense Out of Life Oligator Sentrix GoldenGate GoldenGate Indexing DASL BeadArray Array of Arrays Infinium BeadXpress VeraCode Intelli Hyb iSelect CSPro GenomeStudio Genetic Energy HiSeq HiScan and Eco are registered trademarks or trademarks of Illumina Inc All other brands and names contained herein are the property of their respective owners Eco Real Time PCR System User Guide Table of Contents Chapter 1 OvervieW 1 ipie 2 Real TimePCR 3 Absolute and Relative Guantification 4 Allelic Discrimination and High Resolution Melt 5 Multiplexing Real Time PCR 6 Chapter2 Workflow 7 Eco System WorkfloW
23. a deen ee ees oe do een eh eee eas oan ee ee ee ae es 31 Electromagnetic Compatibility 32 Cleaning And Maintenance aea eee ees 33 sis es oo 4 a4 ve a burs RAS ES DL CARE b 2 k S Rees ta 34 ET 7 Re k pu H a Dino ene pnm dert ai Ca T AT Ga aft e GCGGC ra vagas rcc GA F ii Eco Real Time PCR System User Guide 2 I 49 deyo System Information Components Thermal System Proprietary hollow silver thermal block filled with circulating conductive fluid provides superior temperature control and thermal uniformity across the sample plate Standard Fast protocol performs 40 PCR cycles in approximately 40 minutes Figure 23 Eco Thermal System Motors Silver hollow Thermal Block containing the conductive fluid Stirring Paddles Optical System gt Two LED arrays provide individual sample well excitation gt Four detection filters support almost all PCR chemistries and multiplex detection ROX is optional gt CCD camera acquires high quality data in all wells and filters at each PCR cycle Factory calibrated optics support SYBR Green FAM HEX VIC ROX and Cy5 dyes You can also use other dyes that are compatible with the excitation and emission wavelengths Figure 24 Optical System CCD Camera Filter Slide Green LED Array Blue LED Array 20 Part 15017157 Rev B Lights The Eco System has three indicator lights on the
24. anually Highlight a Standard Assay well and click the Assign button beside the appropriate dilution quantity Figure 16 Eco Real Time PCR System User Guide Figure 15 Selecting Standard Assay Wells Figure 16 Assigning Dilutions Set Up Standards Units Define Standards D ssign Quantity Apply Standards S Apply standards 1200 nd 120 pe T my s 12 S 0 12 dm 0 12 p 012 mecum m fo F S I 0 012 C ROX Normalization 1 moAe7 le q BY dN 18S Set Up the Thermal Profile File Edit Options Help amg Dye7 DNA Standard Curve Drag to Move Stage Plate Layout Thermal Profile Melt Curve Setup Type New Temperature Polymerase Activation UDG Incubation Workflow 100 C 75 C e Setup 50 C Drag Bar Up or Down to 00 15 Adjust Temperature 10 00 Q 02 00 Data Collection Point m Type New Cycle Time FEN Toggle Two Step and mm ci 3 Three Step PCH Number of Cycles 40 2 o Step1 Step2 Estimated Program Length 1 hr 28 mins Delete Add Stage Start Run Click to Add or New Sessioni J Remove Cycles When you define the experiment a corresponding default thermal protile is selected automatically You can use this or modify it based on your reagent recommended protocol You can set up cycle parameters in the Thermal Profile at any time after defining the experiment but before starting the run Click uy to
25. ation about the many possible applications of Eco technology Part 15017157 Rev B Real Time PCR Polymerase Chain Reaction PCR denotes Figure 1 Main Real Time PCR Chemistries the amplification of DNA templates Annealing Annealing catalyzed by DNA polymerase in the L eee O presence of primers dNTPs divalent e eo cations like Mg 2 and a buffer solution e le The ability to visualize and quantify the o O amplification of DNA as it occurs during so pum PCR is called Real Time qPCR or e i fD Quantitative PCR This is made possible gt by the use of fluorescent chemistries an DNA Binding Dyes Hydrolysis Probes optical system that can capture the emitted fluorescence at every PCR cycle and software that can quantify the amplification The two most commonly used qPCR chemistries are DNA binding dyes and hydrolysis probes Figure 1 DNA binding dyes fluoresce when bound to double stranded DNA Hydrolysis probes fluoresce when the reporter molecule is removed from its quencher molecule by the 5 endonuclease activity of DNA polymerase Figure 2 The Three Phases of qPCR Little fluorescence is generated during initial PCR cycles Figure 2 Data from these early cycles define the baseline for the assay Phase I As fluorescence approaches the level of optical detection the reaction reaches the exponential phase Phase ITI which is the region where the Cq is determined Cq is the PCR cycle at wh
26. button appears to the right of the assay role ds Figure 14 Set Up Standards Pane 1 Click Standards to open the Set Up Standards Workflow pane in the lower left of the window i e 2 Select the units that are used in your standards and o Define Standards then enter the dilution information ca O ls Apply Standarde Auto Calculate Serial Dilutions OB o 1 To auto calculate serial dilutions click Def E The Dilutions dialog box opens E Number of Points Starting Quantity Dilution Factor 600 io 7 FA 2 Enter the number of points in the standard curve the quantity of the most concentrated standard and the desired dilution factor and then click Manually Enter Dilutions 1 Enter the value of the first standard into the first Quantity field below Units 2 Press Enter to commit the value and open the next Quantity field 1 6 Part 15017157 Rev B Assign Standard Dilutions to Wells You can assign standard dilutions to wells manually or automatically To assign dilutions automatically Recommended 1 Left click and drag the mouse over a group of Standard Assay wells Vertical Wells Points on Standard Curve Horizontal Wells Replicates The Apply Standards button becomes active when you have selected a suitable group of wells 2 Click priuStandards The dilutions and replicates are automatically added in the highlighted group of wells To assign dilutions m
27. d 3 6 A slope of 3 32 indicates 100 efficiency meaning that the number of template molecules doubled in each PCR cycle Common reference genes High expression 185 ribosomal RNA 185 Beta actin ACTB Beta 2 microglobulin B2M glyceraldehyde 3 phosphate dehydrogenase GAPDH and phosphoglycerokinase PGK Medium expression Transferrin receptor TfR Low expression Transcription factor IID TATA binding protein TBP and glucuronidase GUS 36 Part 15017157 Rev B Glossary a Eco Real Time PCR System User Guide E s of CONATI rnm ardeu carma La tr ff w a G 49Ideyo Glossary 38 Absolute Quantification An assay that quantifies unknown samples by interpolating their quantities from a standard curve based on a serial dilution of a sample containing known concentration Allelic Discrimination An assay that discriminates between two alleles gene variants Amplicon A fragment of DNA synthesized by a pair of primers during PCR Assay The set of primers or primers probe used to quantify an amplicon Baseline The initial PCR cycles when little fluorescence signal is generated This will be used to subtract the background Channel The combination of excitation and emission spectra used to monitor amplification for a given assay Ct Threshold Cycle See Cq Cq Quantification Cycle The cycle number at which the fluorescent signal crosses the threshold It
28. ected within the instrument specifications 25 20 Log of starting quantity target Part 15017157 Rev B Workflow Eco System WorkKflOW RR a 8 ko ehi dad ci hentai 1T I 9 Define a New Experiment LL 10 Set Up the Plate Layout 12 Set Up the Thermal Profile TO MONO Sls saga 3 713239209 AMG ELLA SO 13 5325239225239 929553293254 19 AnalyzeData 20 af af a a ka no Eco Real Time PCR System User Guide I c JajdeyO Workflow Eco System Workflow Quick Start Run Can be done at any time after defining the experiment and before analyzing the results 1 Prepare the sample plate load it into the Eco and close the lid page 9 2 Launch the Eco software on the netbook PC 3 Define the experiment by selecting the application detection chemistry and starting material page 10 v 4 Setup the plate layout by defining assays samples and standards and assigning them to wells page 12 TIP If you wish click Start Run to begin the quick start run now You will be prompted to confirm the thermal profile and name the run Set up the plate layout during the run or any time before analyzing the results 5 Review the thermal profile and adapt it if needed page 18 6 Start the run The software has a tab for monitoring the run in real time page
29. en the primer is incorporated into DNA during PCR the fluorophore is de quenched leading to an increase in fluorescent signal Melt Curve See Derivative Melt Curve Minor Groove Binders MGBs dsDNA binding agents typically attached to the 3 end of Taqman probes MGBs increase the Tm value of probes thus leading to smaller probes Eco Real Time PCR System User Guide 39 Glossary 40 Molecular Beacons Hairpin probes containing a sequence specific loop region flanked by two inverted repeats A quencher dye at one end of the molecule quenches the reported dye at the other end Sequence specific binding leads to hairpin unraveling and fluorescent signal generation Multiplexing Simultaneous analysis of more than one template in the same reaction No Template Control NTC An assay with all necessary components except the template Normalization The use of control genes with a constant expression level to normalize the expression of other genes in templates of variable concentration and quality Passive Reference A fluorescence dye such as ROX that the software uses as an internal reference to normalize the reporter signal during data analysis Peltier Element used for heating and cooling in a qPCR machine Quencher Molecule that absorbs fluorescence emission of a reporter dye when in close proximity TAMRA and BHO are quenchers R Coefficient of Correlation The coefficient of correlation betwee
30. erview Introduction The Eco Real Time PCR System offers life science researchers a full featured real time PCR system at an attractive price Its features include Four color multiplexing gt High Resolution Melt HRM Fast PCR cycling 40 cycle PCR in 40 minutes gt User friendly MIQE compliant software Eco s proprietary technologies provide excellent optical performance along with unmatched temperature control and thermal uniformity for a plate based format 0 1 C Its robust optical system contains two sets of 48 LEDs for a broad range of fluorophores along with four emission filters and a CCD camera for detection enabling multiplexing of up to four targets It is factory calibrated for SYBR Green I dye FAM HEX VIC ROX and Cy5 Eco supports multiple applications including gene expression quantification and analysis and genotyping by allele discrimination or high resolution melt HRM The system includes easy to use data analysis software preloaded on a netbook computer along with other accessories and consumables to provide the best user experience The software is also provided on a USB drive so that it can be installed on additional computers for convenient access To order Eco materials and accessories go to https icom illumina com If you do not have an account yet click Create New User Go to http www illumina com ecoqpcr for Eco resources including tutorials customer stories and inform
31. he mouse over a region of the plot Zoom Out Right click on the plot and select Undo Zoom Select Assays to View Auto Scroll Shows rows corresponding to signal lines Tabular Panel Select rows to highlight corresponding signal lines Graphical Panel Select lines to highlight corresponding table rows Drag Vertical Bar to Resize Panels Font Size b Grid Options b Export Manual Scale Line Width b The zoom controls are the same for all Eco graphs Part 15017157 Rev B Component Data Tab The Component Data tab displays RFU Relative Fluorescent Unit versus cycle number The data displayed are dye deconvoluted to remove dye cross talk when multiplexing Figure 17 Component Data Tab ont eb Component Data fc Amplification Plot Component Data of Melt Curve Above the graph click to view the Amplification Plot or bs to view the Melt Curve Eco Real Time PCR System User Guide 2 1 ejea ezApuv Amplification Plot Tab The Amplification Plot tab displays RFU normalized Rn to the ROX fluorescence level if used versus the cycle number ARn is the Rn normalized to the background Figure 18 Amplification Plot Tab Opera Quarticatorv DNA Binding Dye ONA Standad Curve Wel Table w Q Avett E Selec as Gear Selection Assay Color Slope Y letercept Efficiency R Pk M 2228 33310 mow 939 Workflow Graph Type Select a linear or logarithmic sca
32. ich the fluorescent signal crosses the detection threshold level and is used for quantification Finally as reaction components are consumed and Threshold line amplicons become abundant the generation of PCR Cycles additional fluorescent signal slows down and eventually reaches a reaction plateau Phase III Phase I Phase l Phase lll Fluorescence Delta Rn Baseline Resources Saiki RK Scharf S Faloona F Mullis KB Horn GT Erlich HA Arnheim N 1985 Science 230 1350 1354 Higuchi R Fockler G Dollinger G and Watson R 1993 Biotechnology N Y 11 1026 1030 Eco Real Time PCR System User Guide 3 dod SUN ESA Overview The two primary methods used to quantify nucleic acids by qPCR are the absolute and relative quantification methods The absolute quantification method is based on a standard curve generated from serial dilution of a DNA template of known concentration Figure 3 Quantification of unknown samples is determined by interpolating the sample Cq from the standard curve A standard curve is useful for assay validation The slope of the standard curve measures the efficiency of the assay E 10 1 slope A slope outside the acceptable range slope 3 1 to 3 6 and E value between Absolute and Relative Quantification Figure 3 Five Point 10 Fold Standard Curve 30 Slope 3 32 copy Efficiency 100 R gt 0 99 10 25 copies y 100 copies 20 10 000 15 copies Log of
33. ick PS Samples to open the Samples dialog box Select the number of samples Define a name and color for each sample Click 4 to close the Samples dialog box and return to Plate Layout Proceed to assign assays and samples to wells Figure 12 Samples Dialog Box Samples Humber of Samples EN Sample Name Standard Cell Line 1 m a Cell Line 2 2 Apply Sample Color Part 15017157 Rev B Assign Assays and Samples to Wells Figure 13 Plate Layout Tab Assigning Assays and Samples moAe7 ajeld AY AN PS EvslustioneiataT New Session c Ki Poea po 53 1 For each type of assay and sample left click and drag the mouse to highlight the corresponding wells as shown in Assay Role and Color b gt Columns 1 and 2 of Figure 13 Wells turn Standard Dilution i gold when they are highlighted Sample Name and Color 2 Clickthe Assign button beside any assay or sample name on the left side of the window You can assign up to four isco assays to a well Well with Multiple ASSayS gt u To remove the assay or sample designation highlight the well and click Assign again to toggle the setting off 3 To change the role of a given assay select the corresponding Assay Role from the drop down list Proceed to define standards Eco Real Time PCR System User Guide 1 5 Define Standards When you set an Assay Role to Standard a small orange Chrx Standard Y Sa Standards
34. ied plate adapter for balance Centrifuge the plate at 250 g for 30 seconds Do not spin more than 500 g Verify that there are no air bubbles at the bottom of the wells 9 Open the Eco lid and place the plate on the block aligning the notch against the top left corner WARNING Forcing the plate into any other orientation could damage the instrument WARNING Be careful not to touch the thermal bonnet above the plate It heats to 105 C 221 F when the instrument is turned on and could result in burns 10 Close the Eco lid The heated cover automatically creates a seal around and on top of the plate to prevent evaporation Proceed to define a new experiment Eco Real Time PCR System User Guide 9 ayeld ay peo Workflow Define a New Experiment 10 Figure 9 New Experiment Tab Experiment Type Application Ople Detection Chemistry SA ati Salertal Tem EE ka a Pini Probes q Exa O wu Digest 7 cem Quantitication Method Standard Curve Experiment ame Kisa Series Double click the Eco icon eco On the desktop to open the software The New Experiment tab opens j TIP it If you wish you can re open recent experiments or example runs from the Saved Experiments tab on this screen Select an Application Option Detection Chemistry and Starting Material When you select the application the software automatically configures options for downstream setup and analysis
35. illumina Eco Real Time PCR System User Guide iIACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACT GAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCT ARTGATAACAGTAACACACTE CTGTTAACCTTAAGATTACTTGTTGATCCACTGATTCAACGTACCGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTAC o uns ee AA a aa TACCATTAAGAGCTACCGTCTTCTGT TAACCTTAAGAT TACT TGATCCACT GAT TCAACGTACCGTAA IACGAAAAGAATGATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACT GATT CAACGTACCGTAAAGAT TACT T GATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCA SAAT TGAGAC Hya alla pe e BRE D CCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCAT TAAGAGCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACC CGAAAATAACCTTAA CT AACGTACTTCTGTTAACCTTAAG AC GTACCG AACGTA n G CGACGAAAAGAAT ACG GAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCT AATGATAACAGTAACACACTTCTGTTAACCTT E TAGES ell Le CCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAACAGTAACACACTTC 3GTACCATTAAGAGCTACCGTGCAACAGTAACACACTTCTGT TAACCT TAAGAT TACT T GAT CCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGA
36. ity Range Two sets of 48 LEDs 452 486 nm and 542 582 nm CCD camera 4 filters Proprietary hollow silver block with Peltier based system EMO 48 well plate 5 20 ul lt 5 minutes Less than 40 minutes for 40 cycles 1 copy Supported resolution to 0 1 C Detection of up to four targets simultaneously four plex Optional ROX 34 5 cm W x 31 cm D x 32 cm H 13 6 in W x 12 2 in D x 12 6 in H 13 6 kg 30 Ib including power supply 120 240 VAC 50 60 Hz 8A Operating 15 C to 30 C 59 F to 86 F Storage 10 C to 38 C 50 F to 100 F Operating 15 90 Relative Humidity Storage 5 95 Relative Humidity Part 15017157 Rev B Symbols gt EC REP F o RE Gt CAUTION Hot Surface SJOQLLI S Do Not Throw in Trash At end of useful life recycle the system or device European Representative Fuse replacement fuses must meet the stated rating Humidity Range on packaging indicates acceptable shipping and storage limits Manufactured By Mark of European Conformity device complies with the EMC Directive 2004 108 EC and the Low Voltage Directive 2006 95 EC Model Number Off On Serial Number Temperature Range on packaging indicates acceptable shipping and storage limits Eco Real Time PCR System User Guide 3 System Information Electromagnetic Compatibility This equipment complies with the emission and immunity requirements described in
37. le Plot Type Toggle y axis between Rn and ARn All Ww Plot Type ARn v by Assay c Graph Type Log w Linear Scale Logarithmic Scale 22 Part 15017157 Rev B Baseline Correction Baseline Subtraction is based on background fluorescence detected between cycles 3 and 15 by default You can adjust the baseline graphically numerically or by using a built in algorithm that sets the best baseline for each well based on the unique profile of their amplification plot To set the baseline automatically Recommended 1 Click a Analysis Settings in the right panel The a a a Analysis Settings dialog box opens T Analysis Settings 2 Select the Auto Baseline check box then click Apply To adjust baseline numerically Perini 1 Click Y Analysis Settings in the right panel The Bo Analysis Settings dialog box opens a O Auto Baseline 2 Enter the desired Baseline Start and Baseline End IS Auto Threshold cycles in the Analysis Settings window then click O O um Apply F777 To adjust baseline graphically i Y Drag diamonds sideways to indicate the interval boundaries Eco Real Time PCR System User Guide 2 3 ejea ezApuv Workflow Threshold Adjustment By default Eco sets a threshold approximately in the exponential growth phase of the amplification curves This threshold can be adjusted manually or automatically to improve the quality of the data Eco performs an algorithm on all amplification
38. n measured Cq values and the DNA concentrations It is a measure of how closely the plotted data points fit the standard curve The closer to 1 the value the better the fit R is ideally gt 0 99 Reference A passive dye or active signal used to normalize experimental results Reference Genes Genes with a wide and constant level of expression Typically used to normalize the expression of other genes Examples of commonly used reference genes 16S 18S GAPDH and b actin Relative Quantification An assay used to measure the expression of a target gene in one sample relative to another sample and normalized to a reference gene Reporter Dye Fluorescent dye used to monitor amplicon accumulation This can be a dsDNA binding dye or a dye attached to a probe Each dye is associated with a certain channel Rn Normalized Reporter Signal Reporter fluorescent signal divided by fluorescence of the passive reference dye ROX carboxy X rhodamine The most commonly used passive reference dye Slope The slope of a standard curve It is a measure of assay efficiency E 10 1 slope 1 where a slope of 3 32 is equal to 10076 efficiency E or an exact doubling of template molecules in each PCR cycle Acceptable efficiencies range from 3 6 90 to 3 1 110 Overly high efficiencies indicate qPCR inhibition usually due to contaminants in the sample Overly low efficiencies typically indicate problems with the reaction mix
39. n the Evaluation Plate buffers extra plates seals or USB drive Tape the box securely for shipment and attach a shipping return label D Part 15017157 Rev B Concepts a Eco Real Time PCR System User Guide que ne T Gary qe c nai m Cars rre ran gt OO tr jm a y 19Ideyo Concepts The weight of one genome g size of genome in bp x 618 g mol bp x Avogadro s number One human genome g 3 x 109 bp x 618 g mol bp x 6 02 x 1023 3 08 x 1012 g One haploid cell sperm egg 3 08 pg of DNA One diploid cell 6 16 pg of DNA There is approximately one copy of every non repeated sequence per 3 08 pg of human DNA The average cell contains 10 20 pg of total RNA About 90 95 of total RNA is rRNA 185 5 85 and 285 1 3 is mRNA RNA concentration ug ul A260 40 D 1000 where D dilution factor and A gt so absorbence at 260 nm DNA concentration ug ul A260 50 D 1000 where D dilution factor and A260 absorbence at 260 nm The exponential amplification of PCR Xn is described by the following equation Xn Xo 1 E n where X number of target molecules at cycle n X initial number of target molecules E efficiency of target amplification and n number of cycles Amplification efficiency E is described by the following equation Ey 10 1 slope E The acceptable range of assay efficiency 90 to 110 or a slope between 3 1 an
40. nd provided on a separate USB drive for installation on additional computers as needed Additional accessories and consumables are provided or available for purchase to ensure the best user experience Use of the Eco for specific intended uses such as polymerase chain reaction PCR Real Time qPCR or high resolution melting HRM may require the user to obtain rights from third parties It is solely the user s responsibility to obtain all rights necessary for the intended use of Eco This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INS
41. nel see following table If they did the data from the assays would be indistinguishable during analysis A red outline around a well indicates that it contains more than one reporter dye from the same channel and requires correction before you can start the run moAe7 9jel d BY dN 18S Channel Excitation nm Emission nm Fluorophores Calibrated on the Eco Reporter 1 452 486 505 545 SYBR Green I FAM 2 542 582 604 644 ROX 5 452 486 562 596 HEX VIC 4 542 582 665 705 Cy5 a If you use ROX as a passive reference for normalization you must use the other three channels for multiplexing 1 On the Plate Layout tab click Q Assays to open the Assays dialog box 2 Select the number of assays For each one e Optional Define a name and color Select a Reporter dye thereby setting the channel If your dye is not listed select a reporter with the same excitation and emission range as your dye see the Channel table above Select a Quencher Quencher molecules absorb fluorescent emissions of reporter dyes when in close proximity By default the quencher is set to None for DNA binding dye chemistry and Non fluorescent for Hydrolysis probes Note that BHO and MGB are considered non fluorescent quenchers 3 Click to close the Assays dialog box and return to Plate Layout Optional Proceed to set up samples Eco Real Time PCR System User Guide 1 3 Workflow Set Up Samples 14 On the Plate Layout tab cl
42. tact Illumina Technical Support Signup for Illumina s monthly Illuminotes newsletter which tells you about new versions of systems or software informational resources and product discontinuations Once a year run a known test sample to confirm accurate analysis CAUTION The Eco system contains materials that may be hazardous to the environment if not disposed of properly Be sure to dispose of materials according to all local state provincial and national regulations Eco Real Time PCR System User Guide 33 ooueuo ure A puy DBulue jo System Information Return Process o4 Follow these directions if you need to return the Eco instrument to Illumina for any reason for repairs for example 1 Follow the instructions in Cleaning And Maintenance on page 33 to clean the instrument Obtain the original shipping box and packaging materials used to ship Eco to you If you do not have them go to http www illumina com ecoqpcr for instructions on ordering return shipping materials Put an empty plate into the instrument for safe shipping and then close the lid A Place the instrument into the white foam packaging ensuring that it is properly positioned for complete protection in shipping B Put the packaged instrument into the shipping box C Package the Dell netbook dock and squeegee into their original boxes and place the boxes on top of the Eco in the shipping box C You do not need to retur
43. ue uoneuiuuuosid DSI Overview Multiplexing Real Time PCR The simultaneous detection of multiple Figure T Examples of Eco Compatible Dyes targets in a single reaction is called multiplexing An advantage of multiplexing is that it permits the inclusion of an internal SYBR Green FAM control assay for normalization purposes significantly increasing data accuracy Another advantage is that multiplexing ROX Texas Red TAMRA conserves sample allowing more data to be obtained from the same amount of material Channel 1 505 545 nm Channel 2 A 604 644 nm Channel 3 A 562 596 nm Validating a multiplex qPCR assay can be HEX JOE TET VIC challenging The advent of more advanced Channel 4 A 665 705 nm qPCR master mixes has significantly reduced the amount of optimization typically Cy5 Quasar 670 required making multiplex qPCR a much more attractive alternative Validation of assays using a standard curve is a must to ensure data accuracy a Factory Calibrated Dyes Figure 8 Standard Curves for Four The Eco Real Time PCR System includes four Multiplexed Assays filter channels Figure 7 which enable detection of up to four separate targets in a single reaction Channel 1 Assay 1 Channel 2 Assay 2 Figure 8 Channel 3 Assay 3 Channel 4 Assay 4 Eco is factory calibrated for certain dyes within each channel marked in Figure 7 but also supports additional dyes that are excited and det
44. ule that absorbs energy at a specific wavelength and emits it back at a different wavelength Fluorescence The immediate release of energy a photon of light as a result of an increase in the electronic state of a photon containing molecule HEX Carboxy 2 4 4 5 7 7 hexachlorofluorescein High Resolution Melt HRM An enhancement of the traditional melt curve analysis which increases the detail and information captured Hybridization Probe A probe that is not hydrolyzed by Taq polymerase Hybridization probes can be used for melt curve analysis Examples include Roche FRET and Molecular Beacons Hydrolysis Probe A probe that is hydrolyzed by the 5 endonuclease activity of Taq polymerase Taqman probes are hydrolysis probes Internal Positive Control IPC An exogenous control added to a multiplex qPCR assay to monitor the presence of inhibitors in the template JOE Carboxy 4 5 dichloro 2 7 dimethoxyfluorescein LED Light Emitting Diode A light that is illuminated by the movement of electrons in a semiconductor material LED lights do not have filaments that burn out and do not get very hot Linear View A view of an amplification plot using linear dRn values y axis versus PCR cycles x axis Log view A view of an amplification plot using log dRn values y axis versus PCR cycles x axis LUX Primer Set A self quenched fluorogenic primer and a corresponding unlabeled primer Wh
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