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1. 4 C Calculate Efficiency if possible from dilution series Global Efficiency Efficiency 12 SE Efficiency 0 05 Use Efficiency of Analyzer AnalyzerMiner v 1 AnalyzerMiner implements the model described by Zhao and Fernald in Zhao and Fernald 2005 PMID 16241897 It operates on the raw fluorescence data v Specify Efficiency for each Detector Use Primer Validation Plate Ls Detector Efficiency SE Eff Plate detector1 2 j 0 05 tr detector2 2 10 05 detector3 2 10 05 detector4 2 0 05 3 Ca Values exist Efficiency Values exist Now the setup is complete and you can press the Analyze button to start the analysis Page 22 30 Stephan Pabinger PCR Tutorial PCR 14 Analysis results 14 1 Overview The page displayed after the analysis has been performed lists the calculated results and the provided legend gives information about the meaning of each result By clicking on one or more Reference Samples you can select the samples used as a reference By clicking on the Show Hide log2 button you can display log2 values of the calculated results Display Normalization Results Experiment SDS experiment Back To Analyze Setup Show Display Bars amp Quality Control Show n oO 3 a a Perform Statistical Test Show Reference Samples sample2 v Save Hormalize Results Seve csv lt Epon Show Hide log2 Lege2. G ddCt reference G SE ddCt reference G SO ddCt reference Is Statistical Reference ddCt SE ddCt SD ddCt 0 023332 0 032996 0 028336 0 040074 Avg replicates 1 0 017272 0 029916 Avg replicates 2 0 025289 0 043801 Page 29 30 Stephan Pabinger PCR Tutorial PCR 16 Export All relevant result can be exported using the provided mechanism In addition each graph can be saved by right clicking on it and selecting e g save image as If you want to save it as SVG file you can use the provided button right beneath the displayed image Export As Sila For more information about the QPCR application please consult the user guide Page 30 30 Stephan Pabinger
3. Page 3 30 Stephan Pabinger PCR Tutorial PCR Next you need to export the component and deltaRn values which hold the values that are needed to analyze the experiment These files will be later uploaded to the QPCR application ABI Prism 7000 SDS Software File1 sds ABT Prism 7000 SDS Software File1 sds le View Tools Instrument Analysis Window Hel S Pl File N S 9 File View Tools Instrument Analwsis Window Help Mew Ctrl h Open Ctrl 0 1 ba E pen r G Plot Y Standard Curve Y gt EE Save es a E Plat Y Standard Curve Y Saye Pe ave fs 4 sample Save Ds H vrae Export Sample Setup Calibration Data Import Sample Setup samples IH nde Export Sample Setup Import Sample Setup Page Setup i l Print Preview Spectra Page Setup Calibration Data Print Ctrl P Componente Print Preview Spectra 1 File1 sds Ge ite Print Ctrl F Component 2 ForElisabethi 24072008 sds EN Delta En Dissociation 1 File1 sds 3 example sds fate 4 Gas EERDER ate SE 27 ForElisabeth24072008 sds N 3 example sds 4 270071108 sds Results sample M vrae sample M vrae sample M vrae sample M vrae 2 2 LC After performing the qPCR experiment the file is exported in the IXO format Select file gt export and save the experiment s ightCycle
4. For more information click on the icon or consult the user guide In this tutorial the standard settings are used Perform delta delta CT calculation Back To Analyze Setup Display Normalization Result Reference Calculation EED samples Samples sample4 sample sample class 1 mE 4 Statistical Test Choose Test Permutation Maan Test 0 TWOSIDED Choose p Value Type Choose Testing Correction K 4 8 Average samples in class You can add as many classes as you want to the statistical test On class acts as the Statistical reference reference class and all other classes are tested for their statistical significant difference to this reference class Do not confuse this with the sample references which are used to reference the samples to a given set of samples no statistical test Each class has a color or pattern associated 1s given a specific name and needs to consist of at least one sample In one class the property Set As Statistical Reference Is set which specifies to which class all other classes are compared In this case the classes consist of 3 biological replicates and are therefore named replicates 1 and replicates 2 Those replicates are then tested for their statistical significant difference Page 26 30 Stephan Pabinger PCR Tutorial PCR Choose Classes Add Class Remove Last Class get s Statistical Reference Green ka sample sampl
5. O Analyzers Successful Error Message no error AnalyzerMiner Page 10 30 Stephan Pabinger PCR Tutorial PCR Run 3 1 List Now you can have a look at the created runs go to Run gt Find Runs Each parsed file is associated with a run which represents a performed qPCR run qPCR experiment The first three symbols are used to inform the user that the run is currently analyzed parsed or deleted By clicking on the gird icon you are linked to the plate layout The chart symbol is a direct link to the charts of the run Experiment Run Run New Run EE Query 76 Edit Display Settings Analyzer Result Parser Results Deleted Runs Results 4 Runs found Page 1of1 Progress Information Upload amp Parse i LT mra a 2 Runs found Page 1 of 1 3 2 Information By clicking on the name of a run information about the used software hardware and instrument setting 1s displayed Moreover this page provides information about the category the used files and the latest successful parsing job Page 11 30 Stephan Pabinger PCR Tutorial PCR 8 3 SDS Displayed is the information page of the SDS run Name at Eb Category Hardware Software Instrument Setting Ls Le Plate Protocol El Description Experiments Create Experiment including plate Export Files i al ENEN Plate Parse Plate File Currently parsing
6. directly to the analysis page and skips the experiment information page Experiment Query F Edit Display Settings Experiments per page 15 25 50 100 1 Experiments found Page 1 of 1 go to page go Nr Name Date na P 1 SDS experiment 2008 09 10 cif 88 X Experiments per page 15 25 50 100 1 Experiments found Page 1 of 1 go to page go By clicking on the name of the experiment you are guided to the following screen which provides links to the runs in the experiment and the calculated Cq efficiency results Page 19 30 Stephan Pabinger PCR Tutorial PCR Show Experiment SOS experiment Date 10 09 2006 tutorial SDS experiment Description Go Show Ct and Efficiency Results Return 13 Analysis To analyze an experiment you have to define several parameters In this tutorial one way to analyze the experiment is shown In order to get detailed information about the parameters please consult the user guide During the analysis relative quantities are calculated using averaging of technical replicates normalization against reference genes and inter run calibration For more details consult the paper gBase relative quantification framework and software for management and automated analysis of real time quantitative PCR data by Hellemans et al 2007 13 1 Cq Calculation Methods Please pick the Cq calculation method you have selected in the user
7. settings Therefore the Cq values for the used runs exist and can be used in the upcoming analysis Analyze Setup Experiment example Show Save Setting Save Fa J Ca Calculation Methods Sample Target Reference Genes Normalization Use Name Description AnalyzerMiner AnalyzerMiner implements the model described by Zhao and Fernald in Zhao and Fernald 2005 PMID 16241897 It operates on the raw fluorescence data and calculates Cq value efficiency and starting O AnalyzerCy0 Efficiency Use the efficiency calculated by another method or determined by primer validation AnalyzerCyO implements the model described by Michele Guescini and Davide Sisti et al in A new O soFARAnalyze AnalyzerSoFar implements the algorithm described hy Wilhelm in Wilhelm 2003 and Wilhelm et al in Wilhelm et al 2003 PMID 12613255 SoFar Stands for lt Software For the Analysis of Real time O SDSAnalyzer AnalyzerSD3 implements an algorithm similar to the one used by by the SDS 2 2 2 software from Applied Biosystems It uses a dynamic baseline created by a line fitted into the area prior to the exponential me Ie me Ki me E me lt Ca Values exist Efficiency Values exist Page 20 30 Stephan Pabinger PCR Tutorial PCR 13 2 Sample Detector Here you can specify which samples and targets are used in the analysis In this tutorial all samples and targets are used Ple
8. 19 written to give you an example of how to use the presented application It shows the typical analysis path which starts with the export of files and ends with print ready charts For this tutorial files of two different vendors are used e Applied Biosystems Abi Prism SDS 7000 abbreviated as SDS e Roche Lightcycler LC 4 05 abbreviated as LC Both files and the generated runs and experiments are available in the QPCR application if you log in as user guest password guest Moreover you can download them from https rtpcr genome tugraz at rtpcr info infoindex html 2 Save and Export Files The first step in the analysis pipeline is to create the files that should be analyzed later on 2 1 SDS After performing the qPCR experiment the file needs to be saved Go to file gt save and save the sds file ABI Prism 7000 SDS Software File1 sds By File View Tools Instrument Analysis Window Help BE 22x Fe File1 sds sample4 sample4 My Recent Documents Desktop sample4 sample4 My Documents t t sample4 sample4 gr My Computer y sample4 sample4 mm o MEEN File name Eie v Places Save as type SDS Documents sds y Cancel 4 sample4 sample4 sample sample2 sample3 sample3 sample4 sample4 sample sample sample2 sample2 sample3 sample3 sample4 sample4 H sample1 sample sample2 sample2 sample3 sample3 sample4 sample4
9. D d _ 20 21 22 detector samnhlaF Sample ar Page 14 30 Stephan Pabinger PCR Tutorial PCR 93 LC was Description Eai Show Display Ct Analyze Results Show Page 1 of 1 Go to page Go tems per page Go Weiner omme Passie reternce Oetecon eona rase reso om g won 9 ore E lose sari me le rea ater sm ven le OCC e er ven la e ste won nc e ean DCI ae oe an ree E ae a uo 1N Pos 4 r Detertar4 mamales L ln knr ds Page 15 30 Stephan Pabinger PCR Tutorial PCR 10 Cq Analyze Results By clicking on the Show button nextto Display Cq Analyze Results an overview page 1s shown which lists the performed Cq analysis results Each result can be exported or displayed in detail shown in the next image Here you can check the calculated Cq and efficiency values Detailed Analyzer Results o _Show detector 26 5125 1 5769 samplee detectar detector 27 9115 1 7334 ear Pd carmel Ata car AE AEO1 168807 6 4574 EE E J mm detector 27 452 1 6970 F m samples detectar 4 305 1 7513 sample4 detector 6 534 1 6009 sample4 detector 6 0422 1 6675 sampleb detectar 0 001 1 7330 F ed sampled Page 16 30 Stephan Pabinger PCR Tutorial PCR 11 Charts 11 1 Information The chart view accessed by clicking on Show nexttoDisplay Charts in th
10. Efficiency Analyzer s Use NTCs in Eq analysis or D LY VI Use HTCs in Hormalization Chart Chart Background AFEFFFF 6 Multiple Parse The Multiple Parse window helps you to parse and analyze your uploaded files It automatically detects all files that have not been parsed and displays them in a list If the export file contains the name of the main file e g SDS gt SDS sds Export gt SDS_component txt it is automatically assigned to the corresponding main file For each file export file combination you can choose whether you want to parse or parse and analyze it By clicking on the submit button the files are sent to the parse and analyze services Using the Progress Information page accessible through a link in the top menu you can keep track of the ongoing processes Page 8 30 Stephan Pabinger PCR Tutorial PCR Multiple Parse Display Files Mot Parsed Update Legend N File Export File 1 Export File 2 Export File 3 Parse Analyze File DeltaRin LUtleExport Top Legend EO N aa marea re per pnns rnn arm ThesaveasDstie Spotedcomponentfie onne arm The savedEDS tie Spode ncucing Sample Setup and AmpiticationData COIE CET II A Lghibveler 20 Tre sevesD00 le ExpotesFluerescece ist over Oe as xL Me OOOO O To view the progress information click on Progress Information in the top menu bar Progress Information The progress information page is u
11. F Mames specified for wells are equal to sample names paces Pase Page 12 30 Stephan Pabinger PCR Tutorial PCR 84 LC Displayed is the information page of the LC run new Instrument setting LCtle rot Description Instrument Setting Experiments Create including plate Ed ET ate Parse Plate File Currently parsing Latest successful parsing job 2008 09 12 LCfle Options sd Mames specified for wells are equal to sample names 9 Plate 9 1 Information The plate view accessed by clicking on Show next to Plate in the run view is used to display general information about the plate barcode description used files and to provide a list showing all wells or capillaries By clicking on a well detailed information about this well is displayed Page 13 30 Stephan Pabinger PCR Tutorial PCR 9 2 SDS Edit Plate ue SS Description SDS File File Filel Dean File1 Component Edit Show Show Display Charts Display Ct Analyze Results ft Design Weiner omea PassveReterene Target eoma Task MO e a same 2 RO e a me EL ME NEE 2 MO o e som no ler RO e samp sima 2 Mo e lener me dl moo o ec oa sano la moo ME NEE IF mooo o e sem same ler EC IEC EC IEC E AE AE e min all Ml om im 50 H m me 2 ere se a me dl 7 AF BRO leem sopa me dl BORO e oa sano ler T FI 27 A11 Mm Ri LC
12. I P sample4 om ample5 6 Repl of Sample H Repl of Sample Fluorescence DO C O K E A oa ie 1 2 3 4 5 E 7 S g 10 12 14 E Repl al samples Samples 9 Repl of Sampled T Benl ei Berle 10 Repl of 5ample5 E n 11 Samples 7 Chart Aus Channel 530 Programs 12 Sample 13 Sampled Fluorescence History 14 Sampled 15 Sample1 U 16 Sample 1 17 Repl of Sampleb 18 Repl of Sample 14 Repl of Samples 0 Repl of Sampled 1 Repl of Sample U 22 Repl of Sample11 Chart Preferences Sample Preferences Load Sample Preferences Save Sample Preferences lear Sample Preferences Fluorescence 530 Print Export 1 3 5 F 42 Copy to clipboard 32 36 40 44 Show legend Select the Data tab and tick XML as the file format Then save the fluorescence values 10 x Picture Data Include IZ Point Index IZ Point Labels e Header seres all HTML Table Delimiter L Excel Tab Filename LCfileE port El Export Cancel Page 5 30 Stephan Pabinger PCR Tutorial PCR 2 3 CSV If the QPCR does not support the files produced by your thermocycler you can upload your results using the generic CSV file format An example file and the corresponding description can be found at Information about the CSV file format For further information consult the user guide 3 Start QPCR and log in After performing the qPCR experiments and exporting the r
13. OPCR Tutorial Version 3 0 Create Date Sep 10 2008 Last modified May 25 2009 by Stephan Pabinger PCR Tutorial PCR Table of content Table oi Conen aae anio 2 II Oe 1 RO OE EE N OR EO EE 3 1 1 OE 3 2 Save and Eport A eS ici 3 2 1 SS PP AE NE N 3 2 HER RE RE ER RE tat pm EE A 2 3 SMA 6 2 Dam OPE Rand oet N o o e ii 6 4 Upload TIES rt Ad 6 Y Pier SEUS aee 7 O EET eT Pas ia 8 1 Piet and ana Zen OOS EE Ee Ge Ie N ee 10 DA a EO RE O OR RE EE NR 11 8 1 IE AE OE AO EE EE OE 11 8 2 MONA Oido ooo N OE 11 8 3 to 12 N O RR O 13 O O PIP EN NE 13 9 1 TETE o oi 13 AE OA 14 9 3 E EEE EE RE EO RE EE OE AE EE 15 10 EgA alyze Resulta 16 11 CAIUS a 17 UE ii dii Ta 0 RE EE RE EE Ee 17 EZ 1 DS EE iaa 17 Ho LE SP O 18 12 SS eME Meroe tate ee 19 13 ADA HTH 20 13 1 Eg Calculation Methods ase SIG Fe GE de RR IE AR Oe ND e Ro 20 15 2 Sample R e aa E 21 Oo AR letfeiER GEES EA DE se Mec E 21 AS Fe am EES AA en ON AN ER 22 14 Analysis TESSA a T 23 ELL E E 23 14 2 Multiple Tiare is nac EE SO DE ER N RE GE DE Re Ge id 25 145 Single atole 24 14 4 Quality Conlara 23 15 Statistica Lost E ON 26 EN O OO 26 H2 21 EE EE ER EE EE EE DOE ET 27 16 EXPO eN aii iii 30 Page 2 30 Stephan Pabinger PCR Tutorial PCR 1 Introduction OPCR is a web application designed for storing parsing managing and analyzing qpCR data Including several different algorithms it can facilitate the analysis of qPCR results 1 1 Purpose This tutorial
14. ar All linear log Export As SVG Page 17 30 Stephan Pabinger PCR Tutorial PCR 11 3 LC Since the Lightcycler 2 0 uses a linear plate layout the grid beneath the chart changed to a list view Rn vs Cycle DeltaRn vs Cycle Rn vs Cycle ay m LE 5 0 7 5 10 0 125 150 175 200 225 Cycle Pos 0 Fos 4 Pos 5 Pos o a T N AA nee Page 18 30 Stephan Pabinger PCR Tutorial PCR 12 Experiment Since the upcoming steps are the same for the SDS and the LC files only the SDS run is considered in the next steps However you can view the results of the LC run by using the guest account of the QPCR application After you have taken a look at the generated runs and evaluated the parsed and analyzed results it is necessary to create a new experiment Experiments consist of one or many runs e g experiment is spread over multiple plates because of the limited amount of wells on one plate which are analyzed together To create an experiment click on Experiment and select New Experiment Define name date and description and choose the runs which should be in this experiment Multiple runs are selected by holding the ct r1 key Experiment New Experiment New Experiment Find Experiments Run Upload amp Parse PCR Management SDS experiment Management 3 tutorial SDS experiment Description LCfile Create Now you see the created experiment The first icon links you
15. ase tick Use Replicate Handling and leave Average technical replicates over plate unticked Cq Calculation Methods Sample Target Reference Genes Normalization Use Replicate Handling Average technical replicates over plates O Samples Used Samples sample sample gt sample3 sample4 sample5 sampleb Targets Used Targets detector detector detector3 detectord detector5 detectorb Ca Values exist Efficiency Values exist 13 3 Reference Genes This tab lets you choose which tragets should act as reference genes It is possible to select multiple reference genes or analyze the experiment without a reference gene Ca Calculation Methods l i Sample Target Reference Genes Normalization Reference Gene s need to be on all plates Reference Gene list Used Reference Genes detector2 detector3 detectord detector5 detectorb detector detector Ca Values exist Efficiency Values exist Page 21 30 Stephan Pabinger PCR Tutorial 13 4 Normalization PCR In this view you can select which efficiency should be incorporated into the analysis To follow the tutorial use Use Efficiency of Analyzer and select the analyzer you have picked in the user settings Ca Calculation Methods Sample Target Reference Genes Normalization Define Efficiency
16. ce genes An example is provided below Please consult the user guide for more information Moreover quality controls are performed for NTCs and technical replicates Multiple Targets l Single Target HE Quality Control target has NTC detector false detector2 false detector3 false detector4 false detector5 false detector6 false detector false detectors false CV M geNorm detector 9 04 0 2565 detector2 8 72 0 2565 Mean 8 88 0 2565 target CDNA detectors sampleb detectors sampled detectors sample4 detectors sample3 detectors sample2 detectors sample1 detector sampleb detector sampled detector sample4 detector sample3 detector sample detector sample detector6 sampleb detector6 sample5 detector sample4 Adataata wl mr dr 7 ExperimentReplicates threshold 0 3 difference 0 1799 2 oaa 0 1712 2 0 2569 2 0 0497 2 0 0477 2 0 2121 2 0 1909 2 0 2016 2 0 1536 2 0 2071 2 2 1 2 2 a 0 0564 0 0 Page 25 30 Stephan Pabinger PCR Tutorial PCR 15 Statistical Test 15 1 Setup Statistical tests are used to test several groups in the software named as class of samples for significant difference between them Here you can define which samples should be included in the test and which samples or which class should act as reference Moreover you can choose the method and which p Value type should be used
17. e samples sampled sampled sampleb Sat AS Statistical Reference sample sample sample4 Add Class Remove Last Class 15 2 Result The upper section of the statistical result page provides links back to the various analysis pages and gives you the opportunity to export the generated results Display Statistical Test Results Bars Page 27 30 Stephan Pabinger OPCR Tutorial OPCR The combined targets view displays the averaged results of each class in this case the classes replicates1 and replicates2 for the selected targets Combined Targets Select detector Targets Fold Change Combined SE 1 0 a G E m E Wd Ad o LL 1 053 1 066 0 955 11B 0 903 detectora detectors E Class 1 m Class 2 detectorl Export As SWG Next the results for each target are shown As an example detector1 is presented in this document Page 28 30 Stephan Pabinger PCR Tutorial PCR Target detector Class a ddCa reference G SE ddCad reference f gt D ddCad reference Is Statistical Reference Class 01887 11214 0 0655 0 0655 0 1134 0 1134 Fold Change detector1 SE 1 0 Fold Change Class 1 Class 2 E sample E samples E sample1 sample4 E samples sample6 Export As SYG In addition to the graphical view results of the statistical test can be displayed in text format This view is accessed by clicking on show next to Display Test Result detector1
18. e Targets Single Target HK Quality Contral A Bar Chant SE 1 0 1 6 detector ore A detectord E detector5 detectorb v Error SE 1 Referentes A Sample E sample2 w Title Group By Sample Display Sampe S detectorl M detector2 detector3 Export As SVG 14 3 Single Target The Single Target tab lets you view the results of a single selected traget It provides the same customizability as the Multiple Target tab and additionally lets you choose the color of each sample and allows you to give each sample an alternative name By using drag and drop you can rearrange the list of the displayed samples In the customize Chart section you can edit the appearance of the chart to your needs The customize chart section lets you additionally adjust the chart Multiple Targets Single Target HE Quality Control Target detector iv Show in title O Bar Chart SE 1 0 1 2 Type NRCq Error SE v 1 x 1 1 References A 10 samplel B sample2 v 0 9 gt Customize Chart Fi C 0 7 opy sort to other targets Export As SVG Page 24 30 Stephan Pabinger PCR Tutorial PCR 14 4 Quality Control Quality control for reference genes can only be performed by selecting multiple referen
19. e plate view is used to display graphs of dissociation and fluorescence data if available To switch between the different views click on the tabs in the top bar Below the chart a grid list is shown which represents the used plate layout By clicking on a well it is included excluded from the chart which is then automatically updated You can select multiple wells at once by holding the ctrl key while selecting them Additionally you can click on every well individually Here you can check whether the melting curve analysis was successful and you can get a rough overview of the shape of the fluorescence curves 11 2 SDS The grid beneath the chart displays the used plate layout It colors empty and omitted wells and currently selected wells are colored in red Dissociation Raw BissociaionDemvate Re Cycle DekaRn vs Cycle Dissociation Derivative di gt m E di a Temperature C AI C4 C5 D4 D5 E4 EX EE aa A E A A A O ee a CAN A A A A A At az ma as as ar as aa ato am az B Bi B Ba Ba T B5 na E Br bs Ba Bio oem ate Cc er ce es ce o ce a cto cm cta p Di oa DS DE w o mo DI mz E EL fe B Ee Er e emo Em ea e are ma ma s e er e a mo m m G a os a os es or ea e om on cz H o m e e re ns He w He a mo m m2 omitted ET Cle
20. ed a Java Plugin Version 1 5 x Dateityp alle Dateien o 2 Using compression is only recommended for remote uploading data that can be well compressed AD Alle Dateien has EE EG eg plain text files xml files and not images or already compressed files Cancel After pressing Upload the files are transmitted to the QPCR application and now available for further analysis steps Multiple File Upload Ey Add Files E Remove Rans retary cee EEN x LCFile ixo Ci Documents and Settings Administ ILEFEExpork xml Ci Documents and Settings Administ 1 EDS sds CDoruments and Settings Administ 1104 S All files were successfully uploaded SD5_Component csv C Documents and Settings Administ EDS DeltaRn cev Ci Documents and Settings Administ 5 Files 1824 Kb to upload left T Compress Files automatic lt Upload Cancel 5 User Settings Before the uploaded files are parsed and analyzed it is necessary to take a look at the user settings which can be found by pressing User Settings in the top bar Here you can select your preferred Cq and Efficiency Analyzers the NTC settings and the chart background color For more information click on the information icon Page 7 30 Stephan Pabinger PCR Tutorial PCR User Settings wtormton J Analyzerdiner Preferred Cq Analyzer s AnalyzerCill gt S0PARAnalyzer AnalvzerMirner AnalyzerHutledGene Lnkeg nalyzer EE IE Es Preferred
21. esults you are ready to use the QPCR application Therefore start your browser e g firefox and go to http rtpcr genome tugraz at Next you have to log in with your provided username and password guest guest RTPCR Database Graz Mozilla Firefox Datei Bearbeiten Ansicht Chronik Lesezeichen Extras Hilfe lt a gt gt gt e Sy Gt 5 https rtpcr genome tugraz at rtperfindex jsp Oy Bioinformatics Graz r E m fi y Home User Guide Information login please login Experiment Run Upload amp Parse PCR Management Z Management mm ad Username guest Password ET Submit System Requirements JavaScript and cookies must be enabled in your browser Screen resolution of at least 1024x768 is strongly recommended Mozilla Firefox All Releases recommended 4 Upload files To upload the created files into the QPCR application go to Upload amp Parse and select New Multiple File Upload Press Add Files and select the newly created files Page 6 30 Stephan Pabinger PCR Tutorial PCR 3 Multiple File Upload Upload amp Parse New File Upload HB Add Files New Multiple Fie Upload Flename rectory Size Kb Find File Upoad Multiple Parse PCR Management Management 0 file s O Kb to upload left 7 Compress Files automatic y Upload Cancel HEN Eer Dateiname xml SDS sds SDS_Component cs S5DS_DeltaRn csw Add Files You ne
22. etector2 Sample 27 7122 0 0279 0 0395 0 1007 0 5253 0 0107 0 0131 0 8994 0 0233 0 0297 0 8994 0 0233 0 0297 sample6 detertor2 Sample 26 6569 0 0173 0 0244 0 0847 0 9113 0 0088 0 0119 0 9062 0 0113 0 0158 0 9062 0 0113 0 0188 sample1 detector3 Sample 33 5452 0 2055 0 2906 0 6126 1 1169 0 1303 0 1842 1 3876 0 1706 0 2412 1 3876 0 1708 0 2412 sample2 detector3 Sample 33 022 0 1097 01551 0 3321 1 465 0 0894 0 1238 0 9334 0 0609 0 0823 0 9334 0 0609 0 0823 sample3 detector3 Sample 33 3122 0 0593 0 0838 0 1779 1 2689 0 0423 0 0596 1 2343 0 0829 0 1171 112343 0 0829 0 1171 eamnlad datarstar3 Carmnlin 39 27947 ANIA METE N NT 14 JERI 1 N NIRO i N NIIT NAIDOO ANARO N ANEGA nadoo nnac N ARGA By clicking on Display Bars amp Quality Control you are directed to the page which graphically displays the analysis results 14 2 Multiple Targets Here you can graphically view the calculated results and compare them for several targets It allows you to customize it in many ways including error type the used sample references the grouping performed in the chart title of the chart and the samples displayed Page 23 30 Stephan Pabinger PCR Tutorial PCR Display Normalization Result Bars Experiment SDS experiment Back To Analyze Setup Show wo Display Normalization Result Perform Statistical Test Show Multipl
23. nd cDNA target task avg Ca SE avg Ca SD avg Ca CV rel Cq SE rel Cq SD rel Cd NRCq SENRCq SDNRCq CNRCq SE CNRCa SD CNRCq sample detector Sample 27 5951 0 1086 0 1536 0 3936 0 793 0 0473 0 0668 0 986 0 07 0 0989 0 986 0 07 0 0989 sample2 detector Sample 26 4899 0 0226 0 0319 0 0852 1 4323 0 0217 0 0276 0 9125 0 0251 0 029 0 9125 0 0251 0 079 sample3 detector Sample 27 2751 0 0299 0 0423 0 1097 0 9453 0 0156 0 0219 0 9195 0 0557 0 0787 0 9195 0 0557 0 0787 sampled detector Sample 26 7381 0 1041 0 1472 0 3892 1 2506 0 0672 0 0949 0 9446 0 0677 0 0957 0 94468 0 0677 0 0957 samples detector Sample 27 9566 0 0451 0 0638 0 1614 0 6494 0 0161 0 0228 1 1118 0 0329 0 0458 1 1118 0 0329 0 0458 sample6 detector Sample 26 9764 0 0253 0 0358 0 0939 1 1097 0 015 0 0212 1 1036 0 0175 0 0246 1 1035 0 0175 0 0246 sample detector Sample 26 8743 0 0948 0 1341 0 3827 0 8157 0 0398 0 0563 1 0142 0 0631 0 0892 1 0142 0 0631 0 0892 sample detector Sample 25 5159 0 0303 0 0429 0 1188 1 7201 0 0747 0 0803 1 0959 0 0538 0 0581 1 0959 0 0538 0 0581 sample3 detector2 Sample 26 2716 0 2141 0 3028 0 8149 1 118 01291 0 1828 1 0875 0 1407 0 1988 1 0875 0 1407 0 1988 sample4 detector Sample 25 8416 0 1476 0 2088 05712 1 4016 0 1097 0 1551 1 0587 0 0969 0137 1 0587 0 0989 0137 samples d
24. pdated automatically Progressinformations per page 15 25 506 100 4 Progressinformations found Page loft go to page go me te memnon pass Rin Je Analzerhiner 45 R parsing AbISDS Parser LightCycler vi EA 100 LCfile LCfile AnalyzerMiner il 10 Filet e AbISDS Parser ABI Y1d1 Ed 100 yd Progressinformations per page 15 25 50 100 4Progressinformations found Page Toft go to page go Page 9 30 Stephan Pabinger PCR Tutorial PCR 7 Parser and analyzer logs After completing the parsing and analyzing processes the results can be view by clicking on New Parser LogandNew Analyzer Login the top menu bar logout User Settings Progress Information New Parser Log New Analyzer Log The parser logs are shown in a list and are colored according to their result The legend provides an explanation for each used color Parser Log Legend ParserResults per page 15 25 50 100 2 ParserResults found Pagedoft ge to page EF ago m bare succosstu ate Ven 2 ParserResults found Page dofd ge to page EE go ParserResults per page 15 25 50 100 Top Color Legend NN Feu Parstngwas aaeei For each analyzed file an Analyzer Log is provided which displays information about the performed analysis Show Cq Efficiency Analyzer Log Plate Name Isopn_20090430_2 Plate 1a 46704 Successful yes Date 14052008
25. r Software 4 05 LCfile 8 File Edit view Tools Window Help D New dis Run Q Analysis 9 Open amp Save FA Report Z Template Sa Run Macro byv gt c N gt aana Ls fae omp Off Instrument Name LC_8020 ID LC_802 Root Y Administral Fe Instrument H Roche H Roche Sel ed System Ad E Experi s Y Prefere Desktop H 1 Specie 1 Templ e My Recent Documents My Documents ra ou My Computer S TOS File name LCfie ixo Places Save as type Object ixo files ixo at Axis Chanmel 530 Programs Fluorescence Hi o fel S O T SN M 1 Repl of Sample e S 19 Repl of Sample8 g 10 S 20 Repl of Sample9 Sg if E 21 Repl of Samplet0 i p HY 22 Repl of Sample11 if M 23 Sample12 u 4 hf ZZ 24 Repl of Samplel2 2 A 10 55 15 38 22 42 29 46 Time h mm 5 Front screen da LCfile Page 4 30 Stephan Pabinger PCR Tutorial PCR In addition the fluorescence values need to be exported Therefore go to Run gt Online Data Display Select all samples choose fluorescence history and select Fluorescence over Cycles Right click on the chart and select Export A Stat Hurn End Program 10 Cycles Color Comp OF Instrument Mame LE 6020 ID LE 8020 Programe Online Data Display Run Notes EEN Aun complete Color Pos Name Current Fluorescence 1 Sample Sample ampled E D
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