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Rotor-Gene Q User Manual - Faculté de médecine de l`université d

1. 8 Ba wild type 100 00 a Noo wild type 99 86 10 Miss heterozygote 98 91 12 Mio heterozygote 96 23 Confidence Percentage Threshold 4 Rotor Gene Q Series Software 1 7 Build 94 Note We recommend cursors are only moved if you wish to avoid areas of the melt curve Movement of the cursors toward the melt phase transitions can affect subtraction plots and confidence percentages The Normalized Graph window displays the normalized melt curves Samples can also be viewed as a difference plot against one of the controls 11 20 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis Rotor Gene Q Series Software high resolution melt demo HRM Normalised Graph Melt A from 82 FarRed Page 1 18 x X File Analysis Run Gain View Window Help Restore XT AUHUNE e S 8 wc New Open Save Start Pause Stop Help Settings Progress Profile Temp Samples Analysis Reports Arrange channels 17 Melt A Farred 17 Mel Alfrom 82 FarRed Page Page 1 1 C0 2 m Ob mn 2 3 a 4 Bank On Bank Off Named On AllOn All Off Edit Samples Normalisation Regions Leading Range Stat 824 2 End 82 8 Normalised Fluorescence D 8 Trailing Ran
2. Rotor Gene Q User Manual 11 2009 7 79 Analysis User Interface 7 8 5 Display Options The display options menu is shown below View Window Help Run Settings i fos d Temperature Graph ds Settings M SE ES S LR Profile Editor Profile Progress Samples Gain Calibration Display Options Kk v Show at Most 2 Analysis Windows v Show at Most 6 Windows Reset All Don t Show This Message Again Dialogs Show at Most 2 If this option is checked a maximum of 2 Analysis analysis windows are shown at once If Windows multiple windows are opened readability may be affected Checking this option closes the first analysis window and replaces it with the last opened window If the option is unchecked more than 2 analysis windows can be displayed Show at Most 6 To improve readability the software Windows removes unused windows when new windows are opened This option is enabled by default as it keeps the Rotor Gene Q software screen clear If it is necessary to see more than 6 windows at once uncheck this option Reset All Don t If this is selected the software will redisplay Show This all dialog boxes where the Do not display Message Again this message again checkbox was Dialogs checked These include messages about suspicious settings that may have previously been set not to display again This may be useful for a new user who is unfamiliar with the Rotor Gene Q or the Rotor Gene Q s
3. E 2008 YE E ii xj E Wamings Operator skipped end of cycle for Cycling Repeat 25 mme Rotor Gene Q Series Software 1 7 Build 94 VIRTUAL MODE Current User CORITOD1 RGQ User 2 RG Operator Run Signatures The audit trail is stored in the Rotor Gene Q run file To avoid any unwanted modification of these files they should be kept in a safe location accessible only by designated Windows accounts However if files are stored in a shared area Run Signatures provide extra security The screenshot shows the Security tab in Run Settings for a file with a Run Signature 7 94 Rotor Gene Q User Manual 11 2009 Analysis User Interface General Machine Options Messages Channels General Information Run Signatures are stored within all newly saved runs These signatures like a wax seal on a document guarantee that no changes have been made outside the software If a file is tampered with the signature becomes invalid Run Signature The signature is valid a The signature for this run file is valid The file contents has not been modified outside of the software The Run Signature is a long word that is generated each time the file is saved and linked to the contents of the file For example the signature for this file is 517587770f3e2 1 72ef9cc9bd0c36c081 If the file is opened in Notepad and an edit is made e g the run date is changed to 3 days earlier the foll
4. Rotor Gene Q User Manual 11 2009 1 15 Safety Information FR Risque d lectrocution Un changement de s lection du voltage ne doit en aucun cas tre effectu pendant que l appareil est branch au secteur Cela peut endommager l appareil et faire sauter les fusibles de s curit Explosive atmosphere W12 The Rotor Gene Q is not designed for use in an explosive atmosphere WARNING DE WARNING Explosionsf hige Atmosph ren Der Rotor Gene Q darf nicht in explosionsf higen Atmosph ren betrieben werden Atmosph re explosive Le Rotor Gene Q n est pas congu pour fonctionner dans une atmosph re explosive Risk of explosion W13 The Rotor Gene Q is intended for use with reagents and substances supplied with QIAGEN kits Use of other reagents and substances may lead to fire or explosion Explosionsgefahr Der Rotor Gene Q ist ausschlie lich mit Reagenzien und Substanzen aus den QIAGEN Kits zu benutzen Die Benutzung von anderen Reagenzien oder Substanzen kann Feuer oder eine Explosion ausl sen Risque d explosion Le Rotor Gene Q a t congu pour l utilisation des r actifs et substances fournis par les kits QIAGEN L utilisation de r actifs et de substances autres que celles indiqu es peut entrainer un risque d incendie ou d explosion Rotor Gene Q User Manual 11 2009 Safety Information WARNING Samples containing infectious agents w14 Some samples used with t
5. To perform EndPoint analysis perform a profile with a hold at 50 C for several minutes then a cycling step with 1 step 50 C for 10 seconds acquiring on the required channel Set the number of repeats to 5 as shown above These times are a guide only and may vary for your particular application The more repeats in the profile the more information available to perform the analysis The analysis will automatically average all the readings to achieve a single value for each sample There is no specific number of repeats required Unless a very high level of accuracy is required 5 repeats are usually sufficient Analysis EndPoint analysis can be performed on a number of channels simultaneously To create a new analysis click on the EndPoint tab select the channels by dragging over them with the mouse pointer and then click Show 7 52 Rotor Gene Q User Manual 11 2009 Analysis User Interface Quantitation Scatter EndPoint Other Cycling Green Cycling A Yellow Show Hide Auto shrink window Define controls When an EndPoint analysis is opened for the first time the following message will be displayed if positive and negative controls have not been defined Rotor Gene Q Series Software k x i To use end point analysis you must have positive and negative controls in each channel To define these controls dick OK Click OK The Edit Samples window appears allowing
6. r Users 5 Administrator E abpc M linsongy r Groups Domain Groups Selected User s Groups RG Analyst RG Operator gt El Administrators Show Advanced Close 4 To assign a group to a user select the user s name from the list The bottom panel will update If the user has no groups they cannot launch the software In the example below we assign the user linsongy to the RG Analyst group by selecting the group on the left hand side then clicking the gt button Groups can be removed by selecting them then clicking the button 7 88 Rotor Gene Q User Manual 11 2009 Analysis User Interface BB User Admin x Current User linsongy r Users Administrator Guest r Groups Domain Groups Selected User s Groups RG Analyst Administrators RG Operator Show Advanced Close 5 Now log in as this user As an RG Analyst the Run menu and Profile button are unavailable However existing files can be opened and analyzed as shown in the screenshot below The status bar indicates that the user CORIT198 linsongy is an RG Analyst Rotor Gene Q User Manual 11 2009 7 89 Analysis User Interface BE Rotor Gene Q Series Software VIRTUAL MODE melt analysis demo EIER File Analysis Gain View Window Help x 7 oe H 9 ww F 2 B B amp R Bless Open Save Help Settings Profile Temp Samples Analysis Reports Arrange Cha
7. 1 10 Symbols on the Rotor Gene Q Location Language Description Near the mains General danger symbol voltage selection before using the switch Rotor Gene Q for the first time make sure the mains setting is correct see Section 4 4 Neben dem DE Allgemeines Schalter f r die Gefahrensymbol Bevor Spannungs der Rotor Gene Q das einstellungen erste Mal benutzt wird vergewissern Sie sich das die Spannungseinstellung korrekt ist siehe Abschnitt 4 4 N A proximit du Symbole g n ral de commutateur de danger avant d utiliser selection du l appareil pour la voltage premi re fois v rifier que la s lection du voltage est correcte voir Section 4 4 Near the sample E Hot surface warning chamber visible when lid is open 1 26 Rotor Gene Q User Manual 11 2009 Safety Information Location _ Language Description In der N he der Warnung vor hei en Probenkammer Oberfl chen Das Symbol ist sichtbar wenn die Haube ge ffnet ist A proximite de la chambre de l appareil Symbole visible lorsque le couvercle est ouvert Type plate on the back of the instrument Attention surfaces br lantes Ben l arri re de ir E Type plate on the back of the instrument Plakette auf der R ckseite des Ger tes Br l arri re de Br Type plate on the back of the instrument Marquage CE Manufacturer Hersteller WEEE mark for Europe Symbol Plakette auf der CE
8. Adjust Scale Auto Scale 250bp Heterozygous 885 BinB 250bp Heterozygous 88 7 Bin B 500bp Wild Type 302 Bin C 80 500bp Wild Type 90 3 Bin C BnB 8855 4 E 500bp Wild Type 302 Bin C Na ESSI THEE Homozygous 80 5 Bin Delles Homozygous 80 3 Bin A ee Settings Import Export Rotor Gene Q Series Software 1 7 Build 94 Mstart A amp 1 Rotor Gene Q series s BEADMEF 407m Peak Calling v Flip sign of dF dT Threshold 030821 z Temperature Threshold E hi Peak Bins Name Value width Bin 8042 4 BinB 98 55 4 New Edit Remove Imported Settings none Import Export 7 38 Rotor Gene Q User Manual 11 2009 Analysis User Interface Flip sign of Before defining peaks ensure that the dF dT dF dT sign is correct for the data set to give positive peaks Defining peaks In melt curve analysis peaks can be defined and reported using different methods One is to automatically call all the peaks for each sample The other is to assign peaks to bins which is useful for genotyping Bins define the area where peaks are expected to occur The melt curve analysis software clusters peaks into bin groups based on actual peak values in the curve Bins can be edited if required Any peak that is within the defined range of the bin will be assigned to the bin If there are 2 bins close together then the pe
9. Cancel 5 Click the Reports button to display the Report Browser Select the analysis with the correct name from the list Click the Show button to display the relative quantitation report The Export option exports the results to a new Excel spreadsheet If a calibrator is included the results are relative to the calibrator sample which has a value of 1 7 36 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 6 5 EB Report Browser Sch m Report Categories General amp Melt Curve Analysis E Quantitation amp Delta Delta CT Relative Quantitation delta delta Relative Quant Analysis amp Comparative Quantitation amp 2 Standard Curves Relative Quantitation Aa xl lates Relative Quant Delta Delta TUN An example of results from this analysis is shown below The C values for the gene of interest GOI CT C values for the normalizing gene Norm CT Delta C Delta Delta Cr and relative concentration Relative Conc are displayed The expression is relative to the calibrator sample which is assigned a relative expression of 1 E Preview sd gt hw Print Save As Email To Word Colour Replicate Name GOI CT GOI Count Norm CT Norm Count Delta CT Delta Delta CT Relative Conc Calibrator 000086 2219 3 19 51 3 2 68 0 00 1 00 Yes 2 72 2 74 2 64 Melt curve analysis Mel
10. Perform Last Run Perform Last Run uses the cycling acquisition and sample definitions from the last run open in the software Three Step with This is a three step cycling profile and a Melt melt curve with data acquisition on the green channel Two Step This is a two step cycling profile with data acquired on green yellow orange and red channels Quenched FRET This is a three step cycling profile and a melt curve Unlike Three Step with Melt acquisition is at the end of the anneal step 6 2 Rotor Gene Q User Manual 11 2009 Operating Procedures Software Nucleic Acid This is a default template for measuring Concentration nucleic acid concentration using Measurement intercalating dyes HRM This folder contains high resolution melt profiles Other Runs This folder contains additional profiles The cycling and acquisition profiles for all templates can be altered using the wizard Note User defined templates can be added to the template list in the Quick Start wizard by copying or saving ret files to C Program Files Rotor Gene Q Software Templates Quick Start Templates After copying a file to this path the template will appear as an icon in the list If you would like custom icons for your templates create a ico image with the same file name as the template Subfolders can be created to group related templates This allows organization of templates which could be convenient if for exam
11. Scaling options To access Adjust Scale click on Adjust Scale at the bottom of the main window or right click on the graph and select Adjust Scale on the menu that appears A scale can be manually entered in the window that appears Rotor Gene Q User Manual 11 2009 8 1 Additional Functions 8 4 8 2 Maximum Dn 4 Minimum D 9 o To access Auto Scale click on Auto Scale at the bottom of the main window or right click on the graph and select Auto Scale on the menu that appears Auto Scale attempts to fit the scale to the maximum and minimum readings in the data To access Default Scale click on Default Scale at the bottom of the main window or right click on the graph and select Default Scale on the menu that appears Default Scale resets the scale to display from O to100 fluorescence units Exporting graphs Picture export The following steps describe how to save an image 1 Right click on the image and select Export from the menu that appears 2 The Export Dialog window appears Select the desired format from the Format list Rotor Gene Q User Manual 11 2009 Additional Functions Export Dialog E Picture Native Data r Forma ia nap as Metafile as PostScript as PDF as PCX as GIF as PNG as JPEG Colors Default Monochrome 3 Select the Size tab and specify the desired size Expor
12. We therefore use the One Sample t Confidence Interval Let U be the mean value for a replicate s Cz values 20 En 1 Then a 100 1 a confidence interval for a C value u is B 2 Rotor Gene Q User Manual 11 2009 Appendix C Appendix C Rotor Gene Q accessories Product Rotor Gene Q 2plex Rotor Gene Q 2plex HRM Rotor Gene Q 5plex Rotor Gene Q 5plex HRM Rotor Gene Q plex Warranty PLUS 1 Basic Rotor Gene Q Warranty PLUS 2 Basic Rotor Gene Q Contents Cat no Real time PCR cycler with 2 channels Inquire green yellow laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler and High Resolution Inquire Melt analyzer with 2 channels green yellow plus HRM channel laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler with 5 channels Inquire green yellow orange red crimson laptop computer software accessories year warranty on parts and labor Real time PCR cycler and High Resolution Inquire Melt analyzer with 5 channels green yellow orange red crimson plus HRM channel laptop computer software accessories 1 year warranty on parts and labor Real time PCR cycler with 6 channels Inquire blue green yellow orange red crimson including laptop computer software accessories 1 year warranty on parts and labor 2 year warranty all labor travel and 9241779
13. 11 1 Instrumentation The Rotor Gene Q provides the following demanding real time and thermo optical capabilities required for HRM High intensity illumination Highly sensitive optical detection Fast data acquisition Finely controlled sample temperature Minimal sample to sample thermal and optical variation 11 2 Chemistry QIAGEN offers the Type it HRM PCR Kit for analysis of SNPs and mutations using HRM and the EpiTect HRM PCR Kit for methylation analysis using HRM available soon Both kits contain the third generation intercalating fluorescent dye EvaGreen The kits combine optimized HRM buffer and HotStarTaq Plus to avoid unspecific amplification products and provide reliable results 11 3 SNP genotyping example In the example shown the Type it HRM PCR Kit was used in HRM analysis to differentiate between homozygous wild type homozygous mutant and heterozygous forms of the human SNP rs60031 276 For technical details consult the Type it HRM PCR Handbook White H and Potts G 2006 Mutation scanning by high resolution melt analysis Evaluation of Rotor Gene 6000 Corbett Life Science HR 1 and 384 well LightScanner Idaho Technology National Genetics Reference Laboratory Wessex Rotor Gene Q User Manual 11 2009 11 3 High Resolution Melt Analysis d d B B Heterozygote Homozygote mutant 8 B i Homozygote mutant Heterozygote 11 4 Rotor Gene Q User Manua
14. Signal level Genotype Rotor Gene Q User Manual Analysis User Interface The threshold is a signal level above which a sample is said to be positive amplified This setting must be adjusted by the user for each run A percentage of fluorescent signal normalized so that the highest signal of the positive controls is 10096 and the lowest signal of the negative controls is 096 An interpretation of different permutations of reactions on different channels For example the genotype heterozygous could be assigned to samples that reacted in both channels green and yellow The genotype can also be used to report the results of reactions with internal controls For example results could be reported as inhibited positive or negative depending on whether a reaction was seen in certain channels or not 11 2009 7 51 Analysis User Interface Profile configuration 1 Roter Selection 2 Confirm Profile M MW 9 New Open Save As Help The run will take approximately 6 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after Insert before Remove This cycle repeats LS time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle 10 seconds Acquiring to Cycling A on Green TI Long Range Touchdown 50 deg for 10 secs Back Save Template Start Run
15. Adjust Scale See Section 8 3 Autoscale Revert to Default Scale Export This saves the graph in a variety of formats see Section 8 4 Copy Chartto This copies the graph image to the Clipboard clipboard Edit Chart in This opens the graph directly in TeeChart TeeChart Office for editing see Section 8 4 Office Print This prints the graph Digital Filter This modifies the currently selected digital filter on the graph The digital filter smoothes data using a sliding window of points 8 6 Rotor Gene Q User Manual 11 2009 8 6 Additional Functions Select digital filter for graph x Show Pinpointer This opens a window that displays the exact coordinates of the mouse pointer position Grouping This visually groups samples that have identical names This can be useful on full rotor runs Selecting this option does not affect calculated values Selected area options An area of a graph can be selected by clicking and holding the left mouse button and dragging the mouse pointer The following options appear Select Only These Samples Deselect These Samples Zoom Cancel Select Only Samples outside the selected area are These Samples deselected Deselect These All samples in the selected area are Samples deselected Zoom This zooms in on the selected area of the graph Click the Default Scale button to zoom out Rotor Gene Q User Manual 11 2009 8 7 Additi
16. Double click on the channel to analyze The melt curves from the raw channel are normalized by averaging all starting and ending fluorescence values and then forcing the end points of each sample to be the same as the average Rotor Gene Q User Manual 11 2009 7 61 Analysis User Interface 7 7 7 7 1 BErotor Gene Q Series Software VIRTUAL MODE high resolution melt demo File Analysis Run Gain View Window Help AGCdPHHiog O z B WD B New Open Save Start Pause Stop Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Melt A FarRed Melt Alfrom 82 FarRed Fi HRM Normalised Graph Melt A from 82 FarRed Page 1 a oj x Page Page 1 Normalised Graph Difference Graph o o I Normalised Fluorescence 825 830 835 640 845 850 855 660 865 870 875 680 885 890 895 deg r Normalisation Regions mutation 100 00 Leading Range E heterozygote 100 00 Start 82 4 mutation 3 72 End 82 8 heterozygote 97 58 heterozygote 97 80 Trailing Range wild type 100 00 Start 833 wild type 99 86 E heterozygote 98 91 End 37 heterozygote 96 23 wild type 99 23 r Confidence Percentage ad hina a7 Fa Threshold 30 Rotor Gene Q Series Software 1 7 Build 94 VIRTUAL MODE 7 Autocalling of samples is achieved by clicking Genotypes Enter the name of the genotype followed by the sample number whic
17. Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 6 Analysis menu 7 6 1 Analysis After clicking Analysis the Analysis window appears This window allows creation of new analyses and display of existing analyses The method of analysis is selected using the tabs A list of the channels that can be analyzed using the selected method is shown Multiple assays run in the same channel can be analyzed independently provided they have been set up as separate pages in the Edit Samples window Pages that have already been analyzed have a green checkmark next to them This means that threshold and normalization settings have been saved for this analysis To view or analyze a channel double click on it The specific analysis window appears CEE x 2 Sid Curves Rel Otrer f SIT Giuantitaton Melt Delta Delta ZT Relative Quantitation E elen EE Allelic Discrininatior Cycling A Greer Comparative Quantitation Cycling A Orance Scatter Graph Analysis Cycling A Red EndPoint Aralysis Cycling Y ellow Concentration Analysis High Resolucion Mek Analysis Show All Analysis Options Show Hide Auto shrirk window Auto shrink Selecting Auto shrink window shrinks the window window when it is not in use Moving the cursor over the window enlarges the window again Rotor Gene Q User Manual 11 2009 7 11 Analysis User Interface 7 6 2 Organizing the workspace Each time a new analysis is start
18. This tab displays messages indicating if the user has made changes such as pausing the cycler or skipping cycles during a run It also displays warnings received during the run This tab should be checked if results are not as expected Channels If configuring a new run the channels tab displays the current configuration of the available channels If viewing an existing run the information displayed represents the configuration of the channels when the run was performed If a run corrupts the channel settings default channels can be restored by clicking Reset Defaults Rotor Gene Q User Manual 11 2009 7 65 Analysis User Interface General Machine Options Messages Channels Tube Layout Security Name Source Detector Gain Create New Name Source Detector Gain Create New 10 Edt Remove Reset Defaults DK Cancel Apply This is the name of the channel This specifies the excitation wavelength of the source LED This specifies the detection wavelength and filter type nm band pass hp high pass This specifies the gain for that particular channel This feature enables creation of new channels Clicking Create New opens a window that asks for a new name source and detection filter The filters can be chosen using the drop down menu next to each window 7 66 Rotor Gene Q User Manual 11 2009 Analysis User Interface Channels Green yello
19. High Resolution Melt Analysis This page intentionally left blank 11 24 Rotor Gene Q User Manual 11 2009 12 12 1 12 2 Troubleshooting Troubleshooting Log Archives The software keeps an unmodified record of each run along with diagnostic information in its Log Archive repository By using the Help Send Support Email option you can send an e mail along with all the necessary diagnostic information to QIAGEN Technical Service see Section 7 12 1 To save disk space only Log Archives of the 60 most recent runs are stored Older run Log Archives will be overwritten as new run log archives are created Regional settings in Windows 98 On some computers the regional settings are configured in a contradictory manner that causes a conflict within the Rotor Gene Q system The problem occurs when different decimal settings are used for currencies and numbers For example if numbers are displayed in a 123 456 789 format but currencies are shown as 299 192 20 then there is a conflict since the number 20 123 is ambiguous On Windows 98 machines this prevents the Rotor Gene Q software from correctly interpreting numbers This problem does not occur with Windows 2000 To correct this setting follow the following steps The names of these options may be different in your system s language 1 Click on the Start Menu Click on Settings Click on Control Panel Double click on Regional Settings Double click on R
20. Orange 585nm BlOnm ROX Redmond Bed alexa568 626nm 660nm Cy5 Quasar670 LCRede40 Crimson 710hp Quasar705 LERed705 alexa680 EE 8 Set HRM run conditions Modify the conditions to suit the amplicon For the first set of experiments allow for a wide melt domain Use the theoretical T as a guide to a suitable range Once you have determined where the product will melt reduce the melt domain to no greater than 10 C Ensure that the start of the melt occurs 5 C prior to the first melt transition The default ramp is set to 0 1 C with a hold of 2 seconds at each step The minimum ramp transition is 0 05 C with a second hold at each step Data is automatically acquired to the HRM channel Automatic Gain Optimisation is performed by default The software will search for the optimal gain setting so that the highest fluorescence value reported is no greater than 70 units on a scale of 100 Note that this can be increased to a maximum of 100 11 14 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis x H 9 New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after Cyclin Insert before Remove Ramp from 70 degrees to 99 a degrees Rising by fo E degree s each step Wait for ET seconds of pre melt conditioning on first step W
21. The Dye Channel Selection Chart helps the user to decide which channel is appropriate for dye they intend to use The dyes shown in the table are those that are commonly used and do not indicate the limits of the instrument The acquisition options described above also apply to Melt steps except that it is not possible to append acquisition data using the Same as Previous menu Melt and hybridisation A Melt is a ramp between 2 temperatures from a lower to a higher temperature The permitted temperature range is 35 99 C To set up a Melt specify the start temperature the end temperature the temperature increments the length of time to hold at the first acquisition temperature before the ramp is initiated the time each increment is to be held for and the acquisition channels A ramp will be generated between the 2 temperatures If the start temperature is higher than the end temperature the name of the step will change to Hybridisation The Acquiring To option set to Melt A in the screenshot below can be changed by clicking the button The Acquisition window will appear and the channels can be selected Rotor Gene Q User Manual 11 2009 6 17 Operating Procedures Software Ramp from o degrees to 30 degrees Rising by m degree s each step Wait for o seconds of pre melt conditioning on first step Wait for p m seconds for each step afterwards Acquire to Melt A on Green When running a
22. This is a conservative statistical measure which can be used as a quality measure This range can be narrowed by running more replicates or by having less variation in the replicates The calculated concentration for all the samples with the same name Note This is not the simple average of the calculated concentrations It is the geometric mean which is a mathematically more appropriate average due to the exponential nature of real time amplification Rotor Gene Q User Manual 11 2009 7 21 Analysis User Interface Rep Calc Conc A range of concentrations that accounts for 95 C l 9596 of the variation in the individual sample as well as the linear regression model on which it is based An interpretation of this measure is that it is the range of concentrations that could be expected 9596 of the time if this run were performed repeatedly with the same amount of variation This is a conservative estimate and the range can be quite large due to the variation inherent in any real time analysis This range can be large if standards are run with concentrations different from the unknown samples if a small number of replicates are used or if there is significant variation IMPORTANT The variations reported by this measure are inherent to the exponential process of real time amplification and are not due to the Rotor Gene Q Similar tests performed on block based cyclers would yield greater variation due to the
23. X Auto G ain Optimisation will read the fluoresence on the inserted sample at Ce different gain levels until it finds one at which the fluorescence levels are acceptable The range of fluorescence you are looking for depends on the chemistry you are performing Set temperature to EU H degrees Optimise All Optimise Acquiring IV Perform Optimisation Before 1st Acquisition Perform Optimisation At 60 Degrees At Beginning Of Run r Channel Settings Green 1 Ir RI Edt Name Tube Position Min Reading Max Reading Min Gain Max Gain DP 10Fl 10 10 Hemove Remove All Manual Close Help Set temperature Before reading the Rotor Gene Q will be 1o Optimise All Optimise Acquiring Perform Optimisation Before Ist Acquisition heated or cooled to match the specified temperature By default this is set as the acquisition temperature Optimise All will attempt to optimize all channels known by the software Optimise Acquiring will only optimize the channels that are used in the thermal profile defined in the run cycling and melt Check this box to perform Gain Optimisation at the first cycle in which data acquisition occurs This is recommended for Auto Gain Optimisation 6 26 Rotor Gene Q User Manual 11 2009 Operating Procedures Software Perform Check this box to perform Gain Optimisation At Optimisation just before starting the run x Degrees
24. and DNA sequence complementarity HRM can be used in genotyping applications such as analysis of insertions deletions or single nucleotide polymorphisms SNPs or to screen for unknown genetic mutations It can also be used in epigenetics applications for detection and analysis of DNA methylation status It can also be used to quantitatively detect a small proportion of variant DNA in a background of wild type sequence at sensitivities approaching 5 This can be used for example to study somatically acquired mutations or changes in the methylation state of CpG islands HRM on the Rotor Gene Q facilitates multiple applications including Identification of candidate predisposition genes Association studies comparing cases and controls genotype to phenotype Determination of allele prevalence within a population or subgroup SNP screening and validation Screening for loss of heterozygosity DNA fingerprinting Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Mutation discovery Determining the ratio of somatic acquired mutations HLA typing HRM is easier and more cost effective than probe based genotyping assays and unlike conventional methods it is a Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis closed tube system that prevents contamination with PCR products Results are comparable to conventional methods such as SSCP DHPLC RFLP and DNA sequencing
25. as doing so may cause damage to the instrument and or blow the fuse 1 3 Environment Operating conditions WARNING AN Explosive atmosphere W12 The Rotor Gene Q is not designed for use in an explosive atmosphere WARNING Risk of explosion W13 The Rotor Gene Q is intended for use with reagents and AN substances supplied with QIAGEN kits Use of other reagents and substances may lead to fire or explosion CAUTION Damage to the instrument C3 Direct sunlight may bleach parts of the instrument and A cause damage to plastic paris The Rotor Gene Q must be located out of direct sunlight 1 4 Biological safety Rotor Gene Q User Manual Specimens and reagents containing materials from biological sources should be treated as potentially infectious Use safe laboratory procedures as outlined in publications such as Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm 11 2009 1 5 Safety Information Samples Samples may contain infectious agents You should be aware of the health hazard presented by such agents and should use store and dispose of such samples according to the required safety regulations WARNING Samples containing infectious agents W14 Some samples used with this instrument may contain infectious agents Handle such samples with the greatest of care and in accordance with the required safety regulations Alw
26. either side of the line because this indicates that the run was not conclusive If the difference between a sample being defined as amplified or not amplified is just a few percent this means that if the reaction were repeated the sample could appear on the other side of the threshold Genotypes This option opens the Genotyping window which is used to define which genotype is detected in each channel Ze Genotyping x Reacting Channels wild Type Cycling A Green Heterozygous Cycling A Green Cycling A Yellow Mutant Cycling A Yellow i This window allows genotypes to be assigned to channels In the example above a sample is heterozygous if readings in Rotor Gene Q User Manual 11 2009 7 57 Analysis User Interface channels Cycling A Green and Cycling A Yellow cross the threshold EndPoint analysis templates EndPoint analysis templates allow the user to export genotype and threshold settings into a single ent file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings none Import Export 7 6 10 Concentration analysis Concentration analysis allows the Rotor Gene Q to be used to measure DNA concentrations or to obtain fluorometer readings The screenshot below shows this analysis E Rotor Gene Q Series Software VIRTUAL MODE DNA concentration DER Fie Analysis Run Gain View Security Window Help ZA EH D IH OQ Ff P B W
27. non transferable immunity from suit to use it with detection by a dsDNA binding dye as described in U S Patents Nos 5 994 056 and 6 171 785 and corresponding patent claims outside the United States for the purchaser s own internal research No real time apparatus or system patent rights or any other patent rights and no right to use this product for any other purpose are conveyed expressly by implication or by estoppel This product Rotor Gene Probe PCR Kit Rotor Gene Probe RT PCR Kit and Type it Fast SNP Probe PCR Kit is an Authorized 5 Nuclease Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under certain patents owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd for using only this amount of the product in the practice of the 5 nuclease process solely for the purchaser s own internal research when used in conjunction with Licensed Probe No right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Purchase of this product Rotor Gene SYBR Green RT PCR Kit and Rotor Gene Probe RT PCR Kit is accompanied by a limited non transferable license under RT and Reverse Tr
28. of increasing It is possible to analyze these data using Quantitation but the Invert Raw Data checkbox should be checked For all other quantitation analysis this option should remain unchecked Invert Raw Data 7 18 Rotor Gene Q User Manual 11 2009 Analysis User Interface Cr Calculation The C value is the cycle number at the point where the amplification curve crosses a threshold of detection By setting a threshold line and calculating the intersection with each of the curves the C value for each sample is established Threshold To set the threshold click on the icon a grid with a red arrow then click and hold on the graph and drag the line to the desired level Alternatively enter a log value Alternatively Auto Find Threshold can be used to automatically determine the threshold When setting a threshold manually it should be set in the exponential phase of the run significantly above the background level to avoid noise and below the onset of signal plateau in later cycles CT Calculation Threshold 0 0353 z Eliminate Cycles no z before Eliminate Cycles To set click on the icon a grid with a red before arrow then click and hold on the graph and drag the line to the right This eliminates the threshold for low cycle numbers Note This is useful when there is noise during the initial cycles for example due to sample mixing effects Rotor Gene Q User Manual 11 2009 7 19
29. parts 3 year warranty all labor travel and 9241780 parts Rotor Gene Q User Manual 11 2009 C 1 Appendix C Product Accessories Rotor Disc 100 Starter Kit Rotor Disc 100 30 Rotor Disc 100 300 Rotor Disc 100 Rotor Rotor Disc 100 Locking Ring Rotor Disc 100 Loading Block Rotor Disc Pipetting Aid Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film 60 Rotor Disc Heat Sealing Film 600 C 2 Contents Kit includes 2 Rotor Disc 100 packs Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film Rotor Disc 100 Rotor and Locking Ring Rotor Disc 100 Loading Block Rotor Disc Pipetting Aid 30 individually wrapped discs for 3000 reactions 10 x 30 individually wrapped discs for 30 000 reactions For holding Rotor Disc 100 discs in the Rotor Gene Q requires Rotor Disc 100 Locking Ring For locking a Rotor Disc 100 in the Rotor Disc 100 Rotor Aluminum block for manual and automated reaction setup in Rotor Disc 100 discs Aid for marking well during manual reaction setup on a Rotor Disc Loading Block Heat sealing instrument for use with Rotor Discs requires Rotor Disc 72 or 100 Loading Block 60 films for sealing Rotor Disc 100 or Rotor Disc 72 discs 10 x 60 films for sealing Rotor Disc 100 or Rotor Disc 72 discs Rotor Gene Q User Manual Cat no Inquire 981311 981313 9018895 9018896 9018909 9018897 9018898 981601 981604 11 2009 Appendix
30. positive and negative controls to be defined To define a sample as a positive or negative control click on the sample type cell then select the relevant control type from the drop down menu Note The controls must be toggled on using the toggler at the right hand side of the main window to perform the analysis Rotor Gene Q User Manual 11 2009 7 53 Analysis User Interface ity Select Positive and Negative Controls x Please select at least one positive and negative control then click OK These controls will be used as references for the fluorescence required for a reaction You should select positive and negative controls for each channel Settings Given Conc Format 122457 Unit RL m Samples E gt Edit Reset Default Gradient B 3i ED ame Type Groups G iven Conc Selected Optical Denaturation Ref Unknown sample 1 untreated Unknown e A7 sample 1 untreated Unknown No 4 sample 1 untreated Unknown No Ab Sample treated Unknown No Ab Sample treated Unknown No A Sample 1 treated Unknown No 48 Sample 2 untreated Unknown No B1 Sample 2 untreated Unknown No BME2 Sample 2 untreated Unknown No Ld B3 sample 2 treated Unknown No es B4 sample 2 treated Unknown No Li B5 sample 2 treated Unknown No L3 BB Sample 3 untreated Unknown No Page Name Page 1 Ed ES New Delete Synchronize pages OK Cancel This screen functions in the same way as the Edit Sa
31. specification Rotor Disc OTV Kit components The following components are required to run an OTV M ARotor Disc OTV Kit which includes m Sealed Rotor Disc 72 OTV Rotor contains TLCs When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Rotor Gene Q User Manual 11 2009 10 1 Optical Temperature Verification 10 3 10 2 W Fluorescent scatter plate insert this insert is black for the Rotor Gene 3000 instrument or white for the Rotor Gene 6000 and Rotor Gene Q instruments m ACD that contains the following files OTV Rotor serial number and expiry date file txt OTV test template file ret Product Sheet pdf factory calibration file rex m Product Sheet Rotor Gene Series Software Version 1 7 or above which contains the easy to use OTV Rotor wizard Rotor Disc 72 Rotor Rotor Disc 72 Locking Ring Running an OTV l Place the fluorescent insert over the emission lens in the bottom of the Rotor Gene Q chamber Place the OTV Rotor Disc into a Rotor Disc 72 Rotor Secure using a Rotor Disc 72 Locking Ring Place the assembly into the Rotor Gene Q and click into place Close the Rotor Gene Q lid Access the Advanced wizard by selecting the Advanced tab in the New Run window In the Advanced wizard click on Instr
32. the sample page that is displayed Pages allow varied independent analyses from one channel data set For example you can run two standard curves in the green channel and generate independent reports More information on setting up sample pages is available in Section 7 8 4 If a 72 Well Rotor is used samples are shown in the format Al to A8 B1 to B8 etc The Toggle Sample ID Display option allows the user to switch to a numerical sample order 1 to 72 Select Non Empty This option deselects any samples that have Samples Select Groups File menu New a Type specified as None in the Edit Samples window This ensures that only samples relevant for the analysis are displayed If you have defined groups this feature will toggle switch on off the display of the samples in the groups Groups are arbitrary collections of samples that allow advanced reporting of statistical results For example groups of treated and untreated patient samples can be defined Groups can be set up in the Edit Samples window After selecting File and then New the New Run window appears This window provides commonly used templates organized under Quick Start and Advanced tabs Once the template is selected the wizard guides you through the run setup and allows modification of settings and profiles 11 2009 7 5 Analysis User Interface x Quick Start Advanced Imports the cyding and ac
33. 10 Gain menu 7 99 7 11 Window menu 7 100 7 12 Help menu 7 100 7 12 1 Send Support E Mail 7 100 8 Additional Functions 8 1 8 1 Analysis templates 8 1 8 2 Opening a second run 8 1 8 3 Scaling options 8 1 8 4 Exporting graphs 8 2 8 5 Spanner wrench icon 8 5 8 6 Selected area options 8 7 9 Maintenance Procedures 9 1 10 Optical Temperature Verification 10 1 10 1 OTV principle 10 1 10 2 Rotor Disc OTV Kit components 10 1 10 3 Running an OTV 10 2 11 High Resolution Melt Analysis 11 1 11 1 Instrumentation 11 3 11 2 Chemistry 11 3 11 3 SNP genotyping example 11 3 11 4 Methylation analysis example 11 5 11 5 Guidelines for successful HRM analysis 11 7 11 6 Sample preparation 11 9 11 7 Software setup 11 9 11 8 Real time PCR data analysis 11 17 Contents 4 Rotor Gene Q User Manual 11 2009 Contents 11 9 HRM data analysis 11 19 12 Troubleshooting 12 1 12 1 Log Archives 12 1 12 2 Regional settings in Windows 98 12 1 12 3 Rotor Gene Q troubleshooting 12 3 12 4 HRM troubleshooting 12 4 13 Glossary 1 Appendix A A 1 Technical data A 1 Environmental conditions A 1 Waste Electrical and Electronic Equipment A 3 Appendix B B 1 Quantitation B 1 Appendix C C 1 Rotor Gene Q accessories C 1 Appendix D D 1 Liability clause D 1 Index Index 1 Rotor Gene Q User Manual 11 2009 Contents 5 Contents This page intentionally left blank Contents 6 Rotor Gene Q User Manual 11 2009 Safety Information 1 Safety Information Before using the
34. 2 Cycling A from 2 Negative Controls 1 Heterozygous Positive Control Het Reaction Reaction 8 Mutation Heterozygous Unknown Het Reaction Reaction 3 Wild type Wild type Unknown Wild Reaction No Reaction Heterozygous Unknown Het Reaction Reaction Heterozygous Unknown Het Reaction Reaction To set the defined controls Wild type Unknown Wild Reaction No Reaction click Edit Samples Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Current User CORBETT linsongy Administrators EndPoint analysis is similar to allelic discrimination in that the results are qualitative and that names can be assigned to certain permutations of reactions over different channels However in EndPoint analysis only a single reading is available in contrast to allelic discrimination which uses a cycle by cycle reading for each sample This means that the user must identify positive and negative controls to facilitate the analysis For the raw data signal levels are normalized relative to the known positive and negative controls for each channel The user then selects a percentage signal level as the threshold Terms used in EndPoint analysis Some terms used in EndPoint analysis are explained below Positive control This is a sample which is known to amplify Negative control This is a sample which is known not to amplify This represents the typical background signal 7 50 Rotor Gene Q User Manual 11 2009 Threshold
35. 3 Edit Profile New Run Wizard window 4 New Run Wizard window 5 Analysis User Interface Workspace Toolbar View raw channels Toggling samples File menu Rotor Gene Q User Manual 4 3 4 3 4 4 4 7 4 8 5 1 5 1 5 4 5 8 6 1 6 1 6 4 6 5 6 6 6 7 6 7 6 9 6 10 6 11 6 12 6 31 6 31 7 1 7 1 7 1 7 1 7 3 7 5 11 2009 Contents 7 5 1 7 5 2 7 5 3 7 5 4 7 6 7 6 1 7 6 2 7 6 3 7 6 4 7 6 5 7 6 6 7 6 7 7 6 8 7 6 9 7 6 10 7 6 11 7 7 7 7 1 7 7 2 7 17 3 7 8 7 8 1 7 8 2 7 8 3 7 8 4 7 8 5 7 9 7 9 1 7 9 2 7 9 3 7 9 4 7 9 5 7 9 6 New Open and Save Reports Setup Analysis menu Analysis Quantitation Two standard curve Delta delta C relative quantitation Melt curve analysis Comparative quantitation Allelic discrimination Scatter graph analysis EndPoint analysis Concentration analysis High Resolution Melt analysis Run menu Start Run Pause Run Stop Run View menu Run Settings Temperature Graph Profile Progress Edit Samples Display Options Security menu Configuration 7 5 7 7 7 9 7 9 7 11 7 11 7 12 7 30 7 34 7 37 7 4 7 44 7 46 7 49 7 58 7 61 7 62 7 62 7 63 7 63 7 63 7 63 7 68 7 69 7 70 7 80 7 8 7 82 Running multiple users on the same computer 7 91 Audit trails Run Signatures Sample locking Locked templates Rotor Gene Q User Manual 11 2009 7 92 7 94 7 96 7 98 Contents 3 Contents 7
36. 3 x 1 7 ml 2x 204374 PCR Kit 400 Rotor Gene Probe PCR Master Mix 2 x 2 ml RNase Free Water Rotor Gene Probe For 400 x 25 ul reactions 3 x 1 7 ml 2x 204574 RT PCR Kit 400 Rotor Gene Probe RT PCR Master Mix 100 ul Rotor Gene RT Mix 2 x 2 ml RNase Free Water Rotor Gene Multiplex For 400 x 25 ul reactions 3 x 1 7 ml 2x 204774 PCR Kit 400 Rotor Gene Multiplex PCR Master Mix 2 x 2 ml RNase Free Water Rotor Gene Multiplex For 400 x 25 ul reactions 3 x 1 7 ml 2x 204974 RT PCR Kit 400 Rotor Gene Multiplex RT PCR Master Mix 100 ul Rotor Gene RT Mix 2 x 2 ml RNase Free Water QuantiTect Virus For 200 x 50 ul reactions QuantiTect 211033 ROX Vial Kit 200 Virus NR Master Mix without ROX dye ROX Dye Solution QuantiTect Virus RT Mix RNase Free Water QuantiTect Nucleic Acid Dilution Buffer QuantiTect Primer For 200 x 50 ul reactions or 400 x 25 ul Varies Assay 200 reactions 10x QuantiTect Primer Assay lyophilized supplied in single tube QuantiTect Rev For 50 x 20 ul reactions gDNA Wipeout 205311 Transcription Kit 50 Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water miScript SYBR Green For 200 reactions QuantiTect SYBR 218073 PCR Kit 200 Green PCR Master Mix miScript Universal Primer Type it Fast SNP For 100 x 25 ul reactions 2x Type it Fast 206042 Probe PCR Kit 100 SNP Probe PCR Master Mix 5x Q Solution RNase Free Water Rotor Gene
37. Analysis User Interface Auto Find This function scans the selected region of Threshold the graph to find a threshold setting which delivers optimal estimates of given concentrations The selected region can be changed by entering new upper and lower bounds in the text boxes which appear For most analyses the default upper and lower bounds are suitable The range of threshold levels is scanned to obtain the best fit of the standard curve based on the samples that have been defined as standards i e where the R value is closest to 1 0 Auto Find Theeshold Results This opens the Quantitation Results window By default this window is opened when an analysis is opened If it has been closed it can be reopened using this command E Quant Results Cycling A Green Page 1 Lo No Name Type Io Given Cone Cop Calc Conc Copie z va Rep Ct Rep Ct Stc Rep Ct 95 Cl Rep Cale Conc fRep Cale Conc 95 1 3108 Standard 1442 30000000 28255064 58 1447 0 10 14 21 14 73 27328521 19019879 38266705 2 3008 Standard 1458 30000000 25142820 16 2 3 108 Standard 1440 30000000 28730050 4 2 4 3007 Standard 17 44 3000000 3420624 141 1748 0 09 17 26 17 69 3327013 2517622 4396613 5 3007 Standard 17 58 3000000 3103391 3 4 6 3007 Standard 17 42 3000000 3467111 156 7 3x06 Standard 2038 300000 285353 43 2098 0 06 20 84 21 13 286528 231158 355162 8 10S Standa
38. At The Rotor Gene Q is heated to the Beginning of specified temperature Gain Optimisation is Run performed and then cycling begins on the first step usually a Denature step This option may be chosen if a Gain Optimisation during the run would result in too much time spent on the initial step Usually Perform Optimisation Before 1st Acquisition is preferred because Gain Optimisation is performed as close as possible to the run conditions Channel This drop down menu allows channels to Settings be added Choose the channel of interest and click Add Edit This opens a window in which the Target Sample Range can be set The Target Sample Range is the range of initial fluorescence that should be set for the sample in the specified tube Auto Gain Optimisation reads each channel using gain settings in the range specified by Acceptable Gain Range It chooses the first gain setting that results in a fluorescence reading within the Target Sample Range In the example shown Auto Gain Optimisation searches for a gain setting between 10 and 10 that gives a reading between 5 and 10 Fl in tube 1 In general for intercalating dyes a Target Sample Range of 1 3 Fl is appropriate while a range of 5 10 Fl is more suitable for probe chemistries Rotor Gene Q User Manual 11 2009 6 27 Operating Procedures Software 6 28 Auto Gain Optimisation Channel Settings r Channel Settings Channel Green Tube Position
39. C Product Contents Cat no Rotor Disc 72 Starter Kit includes 3 Rotor Disc 72 packs Inquire Kit Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film Rotor Disc 72 Rotor and Locking Ring Rotor Disc 72 Loading Block Rotor Disc Pipetting Aid Rotor Disc 72 24 24 individually wrapped discs for 1728 981301 reactions Rotor Disc 72 240 10x 24 individually wrapped discs for 981303 17 280 reactions Rotor Disc 72 Rotor For holding Rotor Disc 72 discs in the 9018899 Rotor Gene Q requires Rotor Disc 72 Locking Ring Rotor Disc 72 For locking a Rotor Disc 72 in the 9018900 Locking Ring Rotor Disc 72 Rotor Rotor Disc 72 Aluminum block for manual and 9018910 Loading Block automated reaction setup in Rotor Disc 72 discs Strip Tubes and 250 strips of 4 tubes and caps for 1000 981103 Caps 0 1 ml 250 reactions Strip Tubes and 10 x 250 strips of 4 tubes and caps for 981106 Caps 0 1 ml 2500 10 000 reactions 72 Well Rotor For holding Strip Tubes and Caps O 1 9018903 ml requires Locking Ring 72 Well Rotor Locking Ring 72 Well For locking Strip Tubes and Caps O 1 9018904 Rotor ml in the 72 Well Rotor Loading Block 72x Aluminum block for manual reaction 9018901 0 1 ml Tubes setup with a single channel pipet in 72 x 0 1 ml tubes Loading Block 72x Aluminum block for reaction setup with 9018902 0 1 ml Multi channel multichannel pipets in 72 x 0 1 ml tubes Rotor Gene Q User Manual 11 2009 C 3 Appendix C Product
40. GmbH No right is conveyed expressly by implication or estoppel for any reagent or kit or under any other patent or patent claims owned by the University of Utah Research Foundation Idaho Technology Inc Roche Diagnostics GmbH or by any other Party This product may be operated only with authorized reagents such as fully licensed QIAGEN kits and assays For further information on purchasing licenses for in vitro diagnostics reagents please contact Roche Molecular Systems 4300 Hacienda Drive Pleasanton CA 94588 and for non diagnostics reagents please contact Roche Diagnostics GmbH Sandhofer Strasse 116 68305 Manheim Germany For applicable countries This real time thermal cycler is licensed under U S Patent No 6 814 934 and corresponding claims in any foreign counterpart patent thereof owned by Applied Biosystems LLC in all fields including research and development all applied fields and human and animal in vitro diagnostics No rights are conveyed expressly by implication or estoppel to any patents on real time methods including but not limited to 5 nuclease assays or to any patent claiming a reagent or kit For further information on purchasing additional rights contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Purchase of this product Rotor Gene SYBR Green PCR Kit Rotor Gene SYBR Green RT PCR Kit and miScript SYBR Green PCR Kit is accompanied by a limited
41. I x D Reports Results dF2 d2C Adjust Scale Auto Scale Default Scale Name Take Off Amplification Comparative Conc Rep Takeoff _ Rep Takeoff 95 CI Rep Amp Rep Amp 95 CI Rep Conc mutation 1 1 00E 00 1 00E 00 Calibra mutation 2 3 04E 01 3 04E 01 mutation 3 S 04E 01 S 04E 01 wild type 1 7 01E 01 7 01E 01 wild type 2 7 38E 01 7 38E 01 wild type 3 7 01E 01 7 01E 01 heterozygote 1 9 04E 01 9 04E 01 heterozygote 2 8 59E 01 8 59E 01 heterozygote 3 9 51E 01 9 51E 01 Comparative quantitation results The reaction efficiency is shown in the Amplification column as a score out of 2 2 100 efficiency Note If you suspect the presence of primer dimers or nonspecific products assess reactions by drawing a derivative plot using the Melt option from the Analysis window Ensure that there is a single peak indicative of a single product If possible run a gel to check that there is a single amplification product If there is more than one product the reaction should be repeated or reoptimized 11 18 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis 11 9 HRM data analysis HRM analysis enables both visual and auto calling of genotypes Results can be viewed as either a normalized melt plot or a difference plot Normalized curves provide the basic representation of the different genotypes based on curve shifting for homozygotes and curve shap
42. Messages Channels Tube Layout seal on a document guarantee that no changes have been made outside the software If a file is tampered with the signature becomes invalid Run Signature The signature is valid The signature for this run file is valid The file contents has not been modified outside of the software DK Cancel Apply Temperature Graph Select Temperature Graph from the View menu or click on the Temp button to bring up the Temperature window The graph shows the temperature of the samples during a run As the run proceeds the Set Actual and Hold time is shown for each step of the program For an existing run file the Temperature window shows the temperature history during the run The vertical scale represents temperature and the horizontal scale represents time Use the scrollbar to scroll backwards and forwards through the Temperature window 7 68 Rotor Gene Q User Manual 11 2009 7 8 3 Analysis User Interface Actual 27 8 Hold For Profile Progress Select Profile Progress from the View menu or click on the Progress button to bring up the Profile Progress window This window shows a graphical representation of the thermal profile associated with the run When performing a run the shaded portion of the window indicates the number of cycles that have been completed There is also an estimate of how many minute
43. Rotor Gene Q it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the instrument in a safe condition The following types of safety information appear throughout this manual WARNING The term WARNING is used to inform you about situations that could result in personal injury to you or other A persons Details about these circumstances are given in a box like this one CAUTION The term CAUTION is used to inform you about situations that could result in damage to the instrument or other A equipment Details about these circumstances are given in a box like this one The advice given in this manual is intended to supplement not supersede the normal safety requirements prevailing in the user s country Rotor Gene Q User Manual 11 2009 1 1 Safety Information 1 1 Proper use WARNING Risk of personal injury and material damage w1 Improper use of the Rotor Gene Q may cause personal AN injuries or damage to the instrument The Rotor Gene Q must only be operated by qualified personnel who have been appropriately trained Servicing of the Rotor Gene Q must only be performed by QIAGEN Field Service Specialists Perform the maintenance as described in Section 9 QIAGEN charges for repairs that are req
44. Sample 4 0 Sample 1 was the positive control with the highest fluorescence so it was set to 10096 The other positive control was slightly lower Sample 4 the lowest negative control was set to O96 It is now clear that sample 5 has probably amplified whereas sample 6 has probably not amplified Note Depending on the positive and negative controls selected it is possible to achieve expression levels of greater than 100 or less than 0 A result of greater than 10096 can be interpreted to mean that the sample is more highly Rotor Gene Q User Manual 11 2009 7 55 Analysis User Interface expressed than the positive controls A result of less than 0 can be interpreted to mean that it is less likely that the sample amplified than that the negative controls amplified Since this analysis is qualitative such results are not a concern If the negative controls result in higher fluorescence than the positive controls the samples have been incorrectly set up and the following message appears EndPoint Analysis Cycling A Green Page 1 g amp Reports Results Genotypes Graph cannot be displayed as the negative controls are either at the same level or higher than the positive controls Normalization in multiple channels It is possible to analyze signal data over multiple channels but the sample setup is more complex EndPoint analysis assumes that multiplexing is performed and so each tube can only hav
45. Stoffe in Elektro und Elektronikger ten R ckseite des Ger tes Marquage RoHS pour la Chine limitation de l utilisation de certaines substances dangereuses dans les quipements lectriques et lectroniques Plaque l arri re de l appareil Rotor Gene Q User Manual 11 2009 1 29 Safety Information This page intentionally left blank 1 30 Rotor Gene Q User Manual 11 2009 Introduction 2 1 Introduction Thank you for choosing the Rotor Gene Q We are confident it will become an integral part of your laboratory Before using the Rotor Gene Q it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the instrument in a safe condition About this user manual This user manual provides information about the Rotor Gene Q in the following sections 1 Safety Information Introduction General Description Installation Procedures Operating Procedures Hardware Operating Procedures Software Analysis User Interface Additional Functions SO CON BDO RA CO WN Maintenance Procedures 10 Optical Temperature Verification 11 High Resolution Melt Analysis 12 Troubleshooting 13 Glossary The appendices contain the following M Technical data M Mathematical techniques M Rotor Gene Q a
46. Technical Services CAUTION Damage to the instrument C4 The Rotor Gene Q must not be used if the lid is broken or A if the lid lock is damaged Make sure that the rotor and locking ring are installed correctly Only use rotors locking rings and consumables designed for use with the Rotor Gene Q Damage caused by use of other consumables will void your warranty WARNING Moving parts W19 In case of breakdown caused by power failure remove the A power cord and wait 10 minutes before attempting to manually open the lid 1 8 Rotor Gene Q User Manual 11 2009 Safety Information WARNING Risk of overheating W20 To ensure proper ventilation maintain a minimum A clearance of 10 cm at the sides and rear of the Rotor Gene Q Slits and openings that ensure the ventilation of the Rotor Gene Q must not be covered 1 8 Heat hazard WARNING Hot surface W21 N The Rotor Gene Q chamber can reach temperatures above 120 C 248 F Avoid touching it when it is hot 1 9 Translations of warnings and cautions This subsection contains translations of the warnings and cautions used in this user manual Each warning or caution has a reference number in square brackets at the top right of its box WARNING The term WARNING is used to inform you about situations that could result in personal injury to you or other A persons Details about these circumstances are given in a box like this
47. Zeichen R ckseite des Ger tes ES Be Rotor Gene Q User Manual 11 2009 1 27 Safety Information Symbol Location Language Description Plakette auf der R ckseite des Ger tes Plaque l arri re de l appareil Type plate on the back of the Instrument mE mE Plakette auf der R ckseite des Ger tes Plaque l arri re de l appareil Type plate on the back of the Instrument Plakette auf der R ckseite des Ger tes Plaque l arri re de l appareil DE FR EN DE FR EN DE FR N Type plate on the back of the Instrument 1 28 Rotor Gene Q User Manual WEEE Zeichen f r Europa Marquage WEEE pour l Europe FCC mark of the United States Federal Communications Commission FCC Markierung der USA Validierung durch die Federal Communications Commission Marquage FCC des Etats Unis validation par la Federal Communications Commission C Tick mark for Australia supplier identification N17965 N15128 C Tick Markierung f r Australien Hersteller Identifikation N17965 N15128 Etiquette C Tick pour l Australie Identification du fournisseur N17965 N15128 RoHS mark for China the restriction of the use of certain hazardous substances in electrical and electronic equipment 11 2009 Safety Information Symbol Location Language Description Plakette auf der DE RoHS Markierung f r China Beschr nkung der Verwendung bestimmter gef hrlicher
48. ainsi que les caract ristiques des fusibles de s curit requis L utilisation de l appareil dans d autres conditions que celles indiqu es par cette plaque n est pas autoris e Electrical hazard W10 Voltage selection may only be changed by your sales distributor or authorized personnel Users should not attempt to change voltage selection Doing so may void the warranty DE FR Gef hrdung durch Elektrizit t Eine nderung der vorgegeben Spannungseinstellung darf nur von autorisierten Personen und QIAGEN Vertriebspartnern durchgef hrt werden Benutzer d rfen die vorgegebene Spannungseinstellung nicht ver ndern Falls dies denoch geschieht erlischt die Ger tegarantie Risque d electrocution Un changement de s lection du voltage ne peut tre effectu que par un personnel autoris ou un distributeur agr L utilisateur de l appareil n est pas autoris changer les param tres de voltage Un changement de ces param tre mettra imm diatement fin la garantie WARNING A DE Electrical hazard w11 Do not change the voltage selector switch setting while the instrument is connected to the mains as doing so may cause damage to the instrument and or blow the fuse Gef hrdung durch Elektrizit t Die Spannungseinstellung darf nicht ge ndert werden w hrend das Ger t an das Stromnetz angeschlossen ist da ansonsten das Ger t Schaden nehmen kann und oder die Sicherung durchbrennt
49. caps A from coming off tubes during an experiment If caps come off during an experiment they may damage the chamber Besch digung des Ger tes Jeder Rotor muss mit einem Spannring locking ring gesichert sein Der Spannring verhindert dass die Probengef e w hrend eines Laufes sich aus dem Rotor l sen k nnen Falls der Spannring nicht verwendet wird k nnen losgel ste Probengef e einen Schaden am Ger t verursachen Deterioration de l appareil Chaque rotor doit amp tre securise par un anneau de serrage locking ring L anneau de serrage vite que des tubes ne quittent le rotor pendant l exp rience Si l anneau de serrage n est pas en place les tubes peuvent quitter le rotor et endommager l appareil CAUTION Damage to the instrument C3 Direct sunlight may bleach parts of the instrument and A cause damage to plastic paris The Rotor Gene Q must be located out of direct sunlight DE Besch digung des Ger tes Direktes Sonnenlicht kann Teile des Ger tes bleichen und Plastikteile sch digen Der Rotor Gene Q darf nicht ins direkte Sonnenlicht gestellt werden D t rioration de l instrument La lumi re directe du soleil peut d colorer des parties de l instrument et endommager des parties en plastique Placer le Rotor Gene Q en dehors de la lumi re directe du soleil 1 24 Rotor Gene Q User Manual 11 2009 Safety Information CAUTION AN DE FR Damage t
50. drives air around chamber Chamber vent opens expelling hot air Centrifugal fan Drives air into chamber Cool air in Illustration of the heating and cooling system 3 2 Rotor Gene Q User Manual 11 2009 General Description 3 2 Optical system With a choice of up to 6 excitation sources and 6 detection filters combined with a short fixed optical path the Rotor Gene Q can be used for multiplex reactions ensuring minimum fluorescence variability between samples and eliminating the need for calibration or compensation Samples are excited from the bottom of the chamber by a light emitting diode Energy is transmitted through the thin walls at the base of the tube Emitted fluorescence passes through emission filters on the side of the chamber and is then collected by a photomultiplier The fixed optical path ensures consistent excitation for every sample which means that there is no need to use a passive internal reference dye such as ROX Reaction Chamber Detection Filters P b e vu Lens vr i RD A PMT Detector Pd Assembly Tubes in Rotor ER Light Spin Past Optics L ses Illustration of the optical system Rotor Gene Q User Manual 11 2009 3 3 General Description Available channels Channel Excitation Detection Examples of nm nm fluorophores detected Blue 365 20 460 20 Marina Blue Edans Bothell Blue Alexa Fluor 350 AMCA X ATTO 390 Green
51. general information about the software including the version of the software and the serial number and model of the instrument The software may be freely copied for use within an organization that owns a Rotor Gene Q The software may not be copied and distributed to others outside the organization 4 7 Updating software Software updates are available from the QIAGEN Web site at www qiagen com Rotor Gene which can be accessed from the Help menu in the software To download the software it is necessary to register online 4 8 Rotor Gene Q User Manual 11 2009 5 1 Operating Procedures Hardware Operating Procedures Hardware This section describes operation of the Rotor Gene Q Rotor types First select which tube type and rotor to use There are 4 rotors available to accommodate different tube types 36 Well Rotor The 36 Well Rotor is red in color The 36 Well Rotor and 36 Well Rotor Locking Ring enable the use of 0 2 ml tubes The tubes do not need to have optically clear caps because the Rotor Gene Q reads fluorescence from the bottom of the tube rather than from the top Domed capped tubes can also be used IMPORTANT Use identical tubes in a run Do not mix different tube types or tubes from different manufacturers as this will affect optical uniformity We recommend use of tubes from QIAGEN which are specially designed for use with the Rotor Gene Q see Appendix C Tubes from alternative manufactu
52. lower temperature uniformity of block based systems To compare cyclers if desired we recommend comparing the standard deviation of the C value Note Each of the columns can be displayed or hidden by right clicking on the window and then selecting or deselecting the column name 7 22 Rotor Gene Q User Manual 11 2009 Analysis User Interface ill Quant Results Cycling A Green Page 1 OF x Given Cone Con Cale Cone a Standard Analysis Standard v No Standard Colour Standard v Name Standard v Type Standard v Ct Standard Standard Standard v Given Conc Copies v Calc Conc Copies v Var wv Rep ct Gtandard v Rep Ct Std Dev Standard v Rep Ct 95 CI Standard v Rep Calc Conc Standard v Rep Calc Conc 8595 CI Standard Standard Standard Standard More detailed information on confidence intervals is available in Appendix B We would like to thank Peter Cook from the Mathematics Department of the University of NSW Sydney Australia whose help was invaluable in verifying the mathematical approaches used For increased convenience the AutoStat feature automatically calculates the average standard deviation and minimum and maximum values of samples of interest Select the results of interest by dragging with the left mouse button and the values are displayed in a table on the right of the screen Rotor Gene Q User Manual 11 2009 7 23 Analysis User Interface In
53. sample is unknown and therefore the gain must be determined to ensure the sample signal is sufficient for detection mm Gain Adjustment x File Help Cycling 4 Green 120 m Temperature 60 Edit Gains Start By default all samples are shown in the display Samples can be removed from or added to the display using the toggler to the right The toggler consists of colored cells each of which corresponds to a sample in the display Samples with a brightly colored cell are displayed while samples with a faded cell are not displayed Samples can be switched on or off by clicking on the cell or by dragging the mouse pointer across several cells at time We recommend performing Manual Gain Adjustment as follows 1 Adjust the temperature in the Manual Gain Adjustment window to the acquisition temperature required for the run Rotor Gene Q User Manual 11 2009 6 29 Operating Procedures Software Note The temperature will not be adjusted while the Rotor Gene Q is operating Restart the Rotor Gene Q to apply changes made to the temperature 2 Click on Start The run begins The Rotor Gene Q temperature is adjusted to the temperature specified in the window The graphs in the window start to display data Wait for the temperature to stabilize 4 Note the end point fluorescence Fl reading If the Fl reading is not at the required level click on Edit Gains and ed
54. see Section 7 8 4 Advanced wizard The Advanced wizard enables options that are not available in the Quick Start wizard such as configuration of gain optimization To use the Advanced wizard select a template by double clicking the template name from the list under the Advanced tab of the New Run window Rotor Gene Q User Manual 11 2009 6 7 Operating Procedures Software Quick Start Advanced Imports the cyding and acquisition and sample definitions Empty Run from the last run open pet in the software erform Last Run EE S Three Step with Melt Two Step Jim d Other Runs Instrument Maintenance eU eS Locked Templates z Ki Open A Template In Another Folder Je Show This Screen When Software Opens Template options provided in this window are similar to those provided when using the Quick Start wizard Section 6 1 Perform Last Run Perform Last Run imports the cycling acquisition and sample definitions from the last run open in the software Empty Run This is an empty run which allows the user to define all parameters of the profile Three Step with This is a three step cycling profile and a Melt melt curve with data acquisition on the green channel 6 8 Rotor Gene Q User Manual 11 2009 Two Step HRM Other Runs Instrument Maintenance Locked Templates Operating Procedures Software This is a two
55. step cycling profile with data acquisition on the green channel only to speed up the run This folder contains 2 high resolution melt profiles This folder contains additional profiles This contains the template used during Optical Temperature Verification OTV For more information see Section 10 This template is locked to ensure the profile will always operate correctly This folder contains templates for QIAGEN kits and assays which cannot be modified by the user see Section 7 9 6 Note User defined templates can be added to the template list by copying or saving ret files to C Program Files Rotor Gene Q Software Templates After copying a file to this path the template will appear as an icon in the list 6 2 1 New Run Wizard window 1 In the next window select the rotor type from the list Check the Locking Ring Attached checkbox and click Next to proceed Rotor Gene Q User Manual 11 2009 6 9 Operating Procedures Software New Run Wizard Welcome to the Advanced Run Wizard Rotor Type 36 Well Rotor 72 Well Rotor Rotor Disc 72 Locking Ring Attached Next gt gt 6 2 2 New Run Wizard window 2 In the next window the user s name and notes about the run can be entered The reaction volume must also be entered If the 72 Well Rotor was selected in window 1 three Sample Layout options are available in the drop down menu 1 2 3 is the default option Most user
56. the calibration hold time This is because the calibration process and denaturation are equivalent in Optical Denature Cycling Changing an existing step to use Optical Denature Cycling To change an existing Denature step in a cycling sequence select the cycle in the list in the Edit Profile window Then select the Denature step by clicking on it in the display 60 deg for 40 secs OK 95 deg for 20 secs Not cquiring Long Range Touchdown Click on the drop down menu and select Optical Denature The temperature and hold time are removed and the Optical Denature icon LX is displayed Rotor Gene Q User Manual 11 2009 6 23 Operating Procedures Software Gain Optimisation When setting up a new run it is helpful to use the Gain Optimisation function This allows you to optimize the gain to a setting that will provide the desired range of starting fluorescence at a set temperature usually the temperature at which data acquisition occurs in each of the channels being acquired The aim of Gain Optimisation is to ensure that all data is collected within the dynamic range of the detector If the gain is too low the signal will be lost in background noise If it is too high all signal will be lost off scale saturated The gain range for each channel is 10 to 10 where 10 is the least sensitive and 10 is the most sensitive When running reactions for the first time we re
57. the window that appears select the Users folder Right click on the right hand window and select New User Local Users and Groups action view amp Bm DR Tree 3 administrator analyst Guest IUSR_NEWLA ran MEM Gmatthew operator testuser MB vusR_NEWL Local Users and Groups Local AY Users CA Groups Refresh Export List Creates a new Local User account View Arrange Icons gt Line up Icons Help Name FulName Rotor Gene analyst Internet Guest Account Internet Guest Account Matthew Herrmann Rotor Gene Operator Rotor Gene Analyst and YSA Server Account Built in account Built in account Built in account Built in account Account For the D gt Enter a username and password By default the user will be created with normal access privileges This means they can run software but not install new programs or change system settings Rotor Gene Q User Manual 11 2009 7 85 Analysis User Interface 6 lt lt Ab xl User name Jrewuser Full name eu User Description o Password Confirm password V User must change password at next logon User cannot change password Password never expires Account is disabled ae Click Create You can now log on as this user Assigning roles to each user You should now assign roles to eac
58. this screenshot the concentrations of several samples are analyzed E Quant Results Cycling A Green Page 1 rf xj Statistics Maximum 28730050 Minimum 25142920 30000000 7 E 30000000 30000000 2 Mean 27328521 3000000 3422624 Std Dev 1 07537 3000000 3103381 Orders of Mag 3000000 3467111 300000 285353 300000 298898 300000 275802 AE Copy KO INAR IMPORTANT The AutoStat feature is context aware This means that where possible it only generates information that is useful For example It is not possible to obtain a 9596 confidence interval from a set of selected calculated concentrations because the regression model must also be taken into account The Orders of Magnitude standard deviation is reported for calculated concentrations rather than an absolute value This is a percentage variation For example a value of 1 07537 represents a 7 5496 variation 278 974 322 611 2 300 000 1 07537 300 000 1 07537 Reporting an absolute value does not make sense for a standard curve The value could be reported at the lowest concentration to create a perceived low error 3 copies or at the high concentration 3 000 000 copies For this reason the Orders of Magnitude standard deviation is reported For calculated concentrations the geometric mean is used instead of the arithmetic mean This accounts for the exponential nature of real time PCR For example in the case of twofold d
59. value will take longer to reach a certain absolute copy number than a sample with a higher amplification value The Rep Conc column of the Comparative Quantitation Results window provides the relative concentration The relative concentration of each sample compared with the calibrator sample is calculated based on the takeoff point and the reaction efficiency This is expressed in scientific notation Note The value displayed in Average Amplification to the right of the represents the standard deviation of the average amplification after removal of outlier amplification Rotor Gene Q User Manual 11 2009 7 43 Analysis User Interface 7 6 7 values If this value is large then there may be a large error in the overall calculated concentration values Relative concentrations are calculated by the software as follows 1 The takeoff point of each sample is calculated by looking at the second derivative peaks 2 The average increase in raw data 4 cycles after the takeoff is calculated This is the amplification value for the sample 3 Outlier amplifications are removed to account for noise in background fluorescence 4 The remaining amplifications are averaged This is the average amplification 5 The average takeoff point is calculated for each calibrator replicate 6 The relative concentration for a sample is calculated as Amplification Calibrator takeoff Sample takeoff 7 The re
60. 006 11 16 36 AM Gain menu Click on the Gain menu to view the Gain Settings for the current run This sets the gain of the specified channel before a run Gain settings are retained from the last run These can be modified if the run has not yet started or in the initial cycles Use the up down arrows next to each text field to modify the fields Then click OK The gain can be changed during the initial cycles A red line will be drawn in the appropriate channel showing where the gain was changed Cycles before the gain change will be excluded from analysis Rotor Gene Q User Manual 11 2009 7 99 Analysis User Interface 7 11 7 12 7 12 1 7 100 Green 10 Yellow 5 Orange 5 m Red 5 m Crimson 7 Window menu This menu enables the windows to be tiled vertically or horizontally or arranged in a cascade Further options are accessible by clicking the arrow on the right of the Arrange button Help menu Under the Help menu Contents will access the Help What s New gives a brief overview of new features added since the previously installed software release Website will open the QIAGEN Web site in a new browser window About This Software provides information about the connected machine the serial number of the Rotor Gene Q and the software version Send Support E Mail The Send Support Email option in the Help menu allows you to send a support email to QIAGEN incl
61. 09 Analysis User Interface Scatter Analysis Graph Cycling A Green Cycling A Yellow Page 1 ild Type Scatter graph Scatter graph analysis templates enable analysis genotype and region settings to be templates exported into a single sct file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings none Import Export 7 6 9 EndPoint analysis EndPoint analysis allows discrimination between amplified and unamplified samples at the end of a run Results are qualitative positive negative not quantitative Rotor Gene Q User Manual 11 2009 7 49 Analysis User Interface EndPoint analysis is shown in the screenshot below E Rotor Gene Q Series Software VIRTUAL MODE allelic discrimination demo Fie Analysis Run Gain View Security Window Help E m EB EE co 2 3 SS ASH Pudi Ff O b B Ww D S lt New Open Save Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Cyding A FAM SYBR Cyding A JOE Cyding A from 40 FAM SYBR 2 Cyding A from 40 JOE E amp Reports Results Genotypes a Signal Level 96 Cycling A from 40 FAM SYBR Circles Cycling A from 40 JOE Diamonds o Threshold 7 Controls Thresholds Positive Controls iti EndPoint Analysis Results Cycling A from 40 FAM SYBR Cycling A from 40 J0E DIS No Name Type Genotype Cycling A ftom
62. 3 Version management This document is the Rotor Gene Q User Manual version 2 0 which incorporates minor updates for Rotor Gene Q instruments produced after November 1 2009 with serial numbers higher than 1109000 and software versions higher than 2 0 0 2 2 Rotor Gene Q User Manual 11 2009 Introduction 2 3 Intended use of the Rotor Gene Q The Rotor Gene Q instrument is designed to perform real time and end point thermal cycling using the polymerase chain reaction PCR and high resolution melting analysis HRM in molecular biology applications as well as for other applications such as concentration measurement protein analysis and enzyme kinetics The Rotor Gene Q if used in combination with QIAGEN Kits indicated for use with the Rotor Gene Q instrument is intended for the applications described in the respective QIAGEN Kit handbooks If the Rotor Gene Q instrument is used with kits other than QIAGEN Kits it is the user s responsibility to validate the performance of such product combination for any particular application The Rotor Gene Q instrument is intended for use by professional users such as technicians and physicians trained in molecular biological techniques and the operation of the Rotor Gene Q instrument Rotor Gene Q User Manual 11 2009 2 3 Introduction This page intentionally left blank 2 4 Rotor Gene Q User Manual 11 2009 Air vents Lid handle Rotor chamber Instrument sta
63. 322 log conc 41 608 Cycling A Orange conce 10 0 309 CT 11 185 CT 3 235 log conc 35 187 Cycling A Bed conce 10 0 316 CT 11 817 CT 3 165 loa conc 37 404 From External Source Post 5 0 Standard Curve Format CT d log Conc Efficiency N A From External Source Pre 6 0 Standard Curve Format D Lo Conc wq CT j Cancel Efficiency N A Help Rotor Gene Q User Manual 11 2009 7 17 Analysis User Interface Using this window a standard curve can be imported from another channel analyzed in the current run or from another run Current Run From Other Run Channels From External Source C Calculation Invert raw data When this option is selected quantitation analyses on other channels from this run are listed with the corresponding standard curves Selecting this option brings up a dialog from which a run file can be selected to open If any quantitation analysis has been performed for the run standard curves are listed for each channel analyzed Note The quantitation analysis settings must have been saved in the run file This lists the analyzed channels and their standard curve formulas In this area M and B values can be entered directly This is useful in cases where the values are from an external source such as an Excel spreadsheet Some chemistries produce a fluorescent signal that decreases exponentially instead
64. 470 10 5105 FAM SYBR Green Fluorescein EvaGreen Alexa Fluor 488 Yellow 53025 557 5 JOE VIC HEX TET CAL Fluor Gold 540 Yakima Yellow Orange 585 5 610 5 ROX CAL Fluor Red 610 Cy 3 5 Texas Red Alexa Fluor 568 Red 625 10 660 10 Cy5 Quasar 670 LightCycler Red640 Alexa Fluor 633 Crimson 680 5 712 high Quasar 705 pass LightCycler Red705 Alexa Fluor 680 High 460 20 510 5 SYBR Green I resolution SYTO 9 LC Green melt LC Green Plus HRM EvaGreen Note For suitable combinations of reporter dyes for multiplexing purposes please refer to the corresponding kit handbooks for example the Rotor Gene Multiplex Handbook or the QuantiTect Virus Handbook 3 4 Rotor Gene Q User Manual 11 2009 Installation Procedures Installation Procedures Requirements Power requirements The Rotor Gene Q operates at M 100 240 V AC at 50 60Hz 520 VA peak Make sure that the voltage rating of the Rotor Gene Q is compatible with the AC voltage available at the installation site Mains supply voltage fluctuations are not to exceed 1096 of nominal supply voltages Grounding requirements To protect operating personnel QIAGEN recommends that the Rotor Gene Q be correctly grounded earthed The instrument is equipped with a 3 conductor AC power cord that when connected to an appropriate AC power outlet grounds earths the instrument To preserve this protection feature do not operate
65. Contents Cat no PCR Tubes 0 2 ml 1000 thin walled tubes for 1000 981005 1000 reactions PCR Tubes 0 2 ml 10x 1000 thin walled tubes for 10 000 981008 10000 reactions 36 Well Rotor For holding PCR Tubes 0 2 ml requires 9018907 36 Well Rotor Locking Ring 36 Well Rotor For locking PCR Tubes 0 2 ml in the 9018906 Locking Ring 36 Well Rotor Loading Block 96 x Aluminum block for manual reaction 9018905 0 2 ml Tubes setup in a standard 8 x 12 array using 96 x 0 2 ml tubes Rotor Disc OTV Kit Kit for optical temperature verification of 981400 Rotor Gene systems includes a Rotor Disc preloaded with thermochromatic liquid crystals fluorescent inserts CD with calibration files requires Rotor Disc 72 Rotor and Locking Ring or Rotor Disc 72 Starter Kit Rotor Holder Metal free standing holder for 9018908 assembling tubes and Rotor Discs into rotors QIAGEN HRM and PCR kits recommended for use with the Rotor Gene Q Rotor Gene SYBR For 400 x 25 ul reactions 3 x 1 7 ml 2x 204074 Green PCR Kit 400 Rotor Gene SYBR Green PCR Master Mix 2 x 2 ml RNase Free Water Rotor Gene SYBR For 400 x 25 ul reactions 3 x 1 7 ml 2x 204174 Green RT PCR Kit 400 Rotor Gene SYBR Green RT PCR Master Mix 100 ul Rotor Gene RT Mix 2 x 2 ml RNase Free Water For information about additional kit sizes visit www giagen com C 4 Rotor Gene Q User Manual 11 2009 Appendix C Product Contents Cat no Rotor Gene Probe For 400 x 25 ul reactions
66. D E c New Open Save Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Cyding A Green i Page Page 1 Ell Concentration Measurement Cycling A Green Page 1 ELS S x D Reports Std Curve Results Av Fluorescence Cycling A Green Circles _NamedOn AlOn AIO Edit Samples 5 00 1 Analysis Method 1900 Linear Regression 000472 3 9 10 1113 14 15 17 18 192122 23 25 26 27 29 30 31 33 34 35 37 38 39 41 424345 4647 Sample Spine Curve Fit 90 Bank On Bank Off Fi Conc Standard Curve Cycling Sele Concentration Analysis Results Sele H No Name Type Given Conc pg41 1 Lambda Standard Standard 1 000 0 2 Lambda Standard Standard 1 000 0 3 Lambda Standard Standard 1 000 0 Lambda Standard Standard 500 0 mancato eso UC Conc Cyeing A Green Page 1 e Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Current User CORBETT linsongy Administrators 7 58 Rotor Gene Q User Manual 11 2009 Analysis User Interface Preparing a run To perform concentration analysis first prepare fluorescent standards and samples ideally in triplicate Preparation of standards A standard curve is used to determine the concentration of DNA from each sample measured The DNA used for the standard curve should be a similar type of DNA as in the samples being measured The concentration of at least one DNA sample should be determined using ultraviolet spectrophot
67. For this reason the gradient is analyzed more frequently than the intercept Main window The main window displays the amplification plots on a log scale Clicking Linear Scale at the bottom of the window changes the scale from the log scale to the linear scale and vice versa Changing between these scales only alters the display of the graphs not the calculations This can be verified by use of the pinpointer tool by right clicking on the graph and selecting Show pinpointer Using a log scale smaller values are more visible on the graph whereas a linear scale facilitates a view of the entire reaction Note Amplification plots update in real time as the Rotor Gene Q is actively acquiring data during a run This real time monitoring of data enables the user to see results as soon as the curves show exponential growth Preliminary conclusions can be drawn and decisions made for the next run Rotor Gene Q User Manual 11 2009 7 29 Analysis User Interface 7 6 3 Quantitation analysis templates Quantitation analysis templates allow the user to export normalization and threshold settings into a single qut file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings none Import Export Two standard curve Relative gene expression analysis using a normalizing gene can be performed using the 2 standard curve method The method requires a stan
68. H Target Sample Range 5 Si Fl up to H 0 FI Acceptable Gain Range 10 to 10 m Cancel Help Remove Remove removes the highlighted Remove All channel Remove All removes all channels Start Start begins Gain Optimisation A gain is chosen which results in fluorescence signal levels within the specified range If fluorescence falls outside the specified range the gain is set to give the closest match possible Manual This opens the Manual Gain Adjustment window see page 6 29 Changing Gain If the gain at the beginning of the run was During a Run too high or too low it can be changed within the first ten cycles A vertical line appears where the gain has been changed The cycles before the change are excluded from the analysis Note Gain Optimisation may chose a setting which does not fall within the specified range This can be due to changes in fluorescence after the first Hold step However the result of Gain Optimisation provides a good indication of the fluorescence level the run will be started on Rotor Gene Q User Manual 11 2009 Operating Procedures Software Manual Gain Adjustment To perform Manual Gain Adjustment click on Manual in the Auto Gain Optimisation Setup window The Manual Gain Adjustment window appears This window displays the fluorescent readings at any given temperature in real time It is used when the background of a
69. November 2009 Rotor Gene Q User Manual Sample amp Assay Technologies Trademarks QIAGEN QlAgility EpiTect HotStarTaq Type it QuantiTect QIAGEN Group HRM Rotor Gene Rotor Disc Corbett Research Pty Ltd Alexa Fluor Marina Blue SYBR SYTO Te xas Red Molecular Probes Inc BLAST US National Library of Medicine CAL Fluor Biosearch Technologies Inc Cy GE Healthcare EvaGreen Biotium Inc FAM JOE ROX TET VIC Applera Corporation or its subsidiaries LC Green Idaho Technology Inc LightCycler Roche Group Microsoft Windows Excel Microsoft Corporation Pentium Intel Corporation Quasar Biosearch Technologies Inc Yakima Yellow Nanogen Inc TeeChartOffice Copyright 2001 2002 by David Berneda All Rights Reserved For applicable countries The purchase of this product includes a limited non transferable license to one or more of U S Patent Nos 6 787 338 7 238 321 7 081 226 6 174 670 6 245 514 6 569 627 6 303 305 6 503 720 5 871 908 6 691 041 7 387 887 7 273 749 7 160 998 U S Patent Application Nos 2003 0224434 and 2006 0019253 and PCT Patent Application No WO 2007 035806 and all continuations and divisionals and corresponding claims in patents and patent applications outside the United States owned by the University of Utah Research Foundation Idaho Technology Inc Evotec Biosystems GmbH and or Roche Diagnostics
70. Q User Manual 11 2009 C 5 Appendix C Product Type it HRM PCR Kit 100 EpiTect MethyLight PCR Kit 200 EpiTect HRM PCR Kit 100 Contents Cat no For 100 x 25 ul reactions 1 x 1 3 ml of 206542 2x HRM PCR Master Mix contains HotStarTaq Plus DNA Polymerase EvaGreen dye optimized concentration of Q Solution dNTPs and MgCl and RNase Free Water Master Mix for methylation specific real 59436 time PCR analysis 200 x 50 ul reactions For 100 reactions 2x EpiTect HRM 59445 Master Mix containing HotStarTaq Plus DNA Polymerase EpiTect HRM PCR Buffer with EvaGreen dye dNTP Mix and RNase Free Water For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor C 6 Rotor Gene Q User Manual 11 2009 Appendix D Appendix D Liability clause QIAGEN shall be released from all obligations under its warranty in the event repairs or modifications are made by persons other than its own personnel except in cases where the Company has given its written consent to perform such repairs or modifications All materials replaced under this warranty will be warranted only for the duration of the original warranty period and in no case beyond the original expiration date of original warranty unles
71. Q software allows the user to export raw data in a format that can then be imported by the LinReg tool for analysis Ramakers C Ruijter J M Deprez R H and Moorman A F 2003 Assumption free analysis of quantitative real time polymerase chain reaction PCR data Neurosci Lett 339 62 7 8 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 5 3 7 5 4 1 Open the Rotor Gene Q run file containing the raw data 2 Export the data to the LinReg export format by selecting Save As and then LinReg Export Format 3 Microsoft Excel automatically displays the exported raw data Start up the LinReg tool The tool asks you to select the cell range where the raw data is located The tool can only analyze one raw channel at a time so an appropriate region of the Excel sheet should be selected Reports After selecting Reports the Report Browser window appears If the data has already been analyzed the report of that analysis can be displayed from the Report Browser window Several report types are offered with varying degrees of detail BE r Report Categories Templates Melt A Green page 1 amp Quantitation Cycling amp Green page 1 amp Delta Delta CT Relative Quantitation delta delta Relative Quant Analysis amp Comparative Quantitation Cycling 4 Green page 1 amp 2 Standard Curves Relative Quantitation 2 standrard curve Relative Quant Anal
72. T method Methods 25 402 7 34 Rotor Gene Q User Manual 11 2009 Analysis User Interface Analysis x 2 Std Curves Rel gr i Seenen uant tation N M t Allele D iscrirination Clearing Poin Analysis Cycling A Green Page Comparative Juantitation wf Cycling Orange Pam Concentration Analysis Delta Delta CT Relative Quantitation EndPoint Analysis High Resolution Melt Analysis Scatter Graph Analysis Show All Analysis Options Show Hide Autoshrink window 3 Enter a name for the analysis Rotor Gene Q Series Software E x Enter a name for the relative quantitation analysis 4 Validation Run Performed must be checked to proceed with the analysis Define the pages where the gene of interest and normalizing gene have been analyzed Rotor Gene Q User Manual 11 2009 7 35 Analysis User Interface Relative Quantitation x Reports Erpat Delta Delta CT Relative Quantitation O Validation Run Performed C Gene of Interest Quantitation oO Normaliser Quantitation U Calibrator Defined Selection of Normaliser Standard Curve zi Currently Selected None To select an existing analysis to use or to create a new analysis select a channel from the list then click Select This window will close and you will be taken to the main window of this analysis e re El Cycling A Orange w Gene of Interest Select Dther Run
73. TUAL MODE Current User CORBETT linsongy Administrators Note If files are e mailed the encryption process can invalidate the signature To avoid this zip the file before e mailing 7 9 5 Sample locking It is important to ensure that sample names are not accidentally or intentionally changed once a user has started a run For this reason the Rotor Gene Q software provides sample locking Sample names can be locked by any user but can only be unlocked by an administrator For users who run their computers in administrator mode this option is of limited value To use this option the computer must be configured securely as described in the previous sections Note If you wish to lock samples do not run the software as an administrator Create an account with RG Operator and RG Analyst groups and keep the administrator password secret Users will then require authorization from the administrator to unlock files 7 96 Rotor Gene Q User Manual 11 2009 Analysis User Interface Samples can be locked before starting a run when using the Advanced wizard by clicking Finish and Lock Samples New Run Wizard Settings Given Conc Format Unit Copies D More ptions Samples Bl Edt Reset Defaut Gradient Die mm Er C ID Name Type Groups Given Conc Se a 1 250bp Unknown Ye 2 250bp Unknown Ye 3 250bp Unknown Ye 4 500bp Unknown Ye 5 500bp Unknown Ye 6 500bp Unknown Ye Unknown Ye U
74. W21 The Rotor Gene Q chamber can reach temperatures of above 120 C 248 F Avoid touching it when it is hot Hei e Oberfl chen In der Reaktionskammer des Rotor Gene Q k nnen Temperaturen von mehr als 120 C 248 F erreicht werden Vermeiden Sie es die Oberfl chen der Kammer zu ber hren solange diese hei sind Surface br lante La chambre du Rotor Gene Q peut atteindre des temp ratures superieures 120 C 248 F Eviter de toucher l int rieur de l appareil quand il est chaud WARNING AN 1 22 Hot surface W22 When pausing a run the Rotor Gene Q will not be cooled completely to room temperature Exercise caution before handling the Rotor or any tubes in the instrument HeiBe Oberfl chen Falls ein Experiment angehalten oder abgebrochen wird wird der Rotor Gene Q nicht automatisch bis auf Raumtemperatur abgek hlt In diesem Fall d rfen die Probengef e und Rotoren in der Reaktionskammer nur mit gr te Vorsicht angefasst werden Rotor Gene Q User Manual 11 2009 Safety Information FR Surface br lante Si une exp rience est suspendue le Rotor Gene Q ne va pas refroidir compl tement jusqu temp rature ambiante Il est alors recommand de faire preuve de pr caution lors du maniement des tubes ou du rotor l int rieur de l appareil CAUTION The term CAUTION is used to inform you about situations that could result in damage to the instrument or other A equipmen
75. a region where a melt peak is expected to occur Genotypes can be defined based on the presence of peaks in certain bins or combinations of bins A channel consists of a light emitting diode LED with an excitation filter paired with an emission filter The LED and excitation filter excite samples at a given wavelength Fluorescence emitted by samples is passed through the emission filter before being detected by a photomultiplier The Rotor Gene Q uses a photomultiplier to collect fluorescence photons and convert them to electronic signals The gain is a setting that determines the sensitivity of the photomultiplier If the gain is set too high the signal is oversaturated If the gain is set too low it is not possible to differentiate signal from background noise Gain Optimisation is a process that dynamically adjusts the gain setting allowing an appropriate setting to be selected which results in optimal signal detection Loading Blocks are aluminum blocks available in different formats which are used to hold tubes or Rotor Discs during reaction setup Rotor Disc Loading Blocks are also used with the Rotor Disc Heat Sealer to heat seal Rotor Discs Locking Rings are metal rings that fit onto the rotor to prevent tubes and caps from coming loose during operation of the Rotor Gene Q Loose caps and tubes could cause damage to the instrument Rotor Gene Q User Manual 11 2009 13 1 Glossary Term Description Rotor The met
76. ace L icon T icon icon E E Icon Icon ES icon Sample Types 7 72 The Open icon brings up a dialog box in which a Rotor Gene Q file can be selected to import Note The number of samples in the open window and the file being imported must match The Save icon brings up a dialog box in which the name and folder can be entered in which a copy of the current sample definitions will be saved The Copy icon copies the selected cells The Paste icon pastes cells that had been selected with the copy command into the currently selected position on the grid The Excel icon brings up a dialog box that prompts for a file name and folder in which to save the sample information After pressing Save the Excel file is opened automatically The Append Overwrite icon changes the editing of cells in the Edit Samples window If overwrite is selected existing data is overwritten when editing If append is selected new data is added to the end of existing data when editing Samples can be defined as one of several types listed in the following table Rotor Gene Q User Manual 11 2009 Analysis User Interface Sample type None NTC Negative Control Positive Control Unknown Standard Calibrator RQ Description No sample in that position No template control Negative control Positive control Unknown sample to be analyzed St
77. ait for 2 seconds for each step afterwards Acquire to HRM A on HRM Gain Optimisation JM Optimise gain before melt on all tubes The gain giving the highest fluorescence less than 70 will be selected md 9 Optional Set Auto Gain Optimisation This applies to the real time amplification step only and is set for the green channel Click the Optimize Acquiring button to optimize only those channels used by a run Optimization is best performed just before the first acquisition step so check the Perform Optimization Before First Acquisition checkbox The recommended background fluorescence range for intercalating dyes is between 1 and 3 Fluorescence units To change this setting click the channel name to select it in the list and then click the Edit button Rotor Gene Q User Manual 11 2009 11 15 High Resolution Melt Analysis Auto Gain Optimisation Setup x r Optimisation Ki Auto Gain Optimisation will read the fluoresence on the inserted sample at e different gain levels until it finds one at which the fluorescence levels are acceptable The range of fluorescence you are looking for depends on the chemistry you are performing Set temperature to ET degrees Optimise All Optimise Acquiring IV Perform Optimisation Before 1st Acquisition Perform Optimisation At 60 Degrees At Beginning Of Run r Channel Settings e dd Name Tube Position Min Reading Ma
78. ak will be assigned to the closest bin Note The bins should not be visually positioned to estimate peak positions Set the bins in the approximate area of interest then use the actual reported values in the results table for a more accurate result Peak Bins To define a bin click the New Bin button then click and hold on the graph to define the center of the bin To add another bin repeat the process Use the Remove button to delete bins Threshold To set the threshold y axis click the hl icon then click and hold on the graph and drag the threshold line to the desired level Rotor Gene Q User Manual 11 2009 7 39 Analysis User Interface Temperature To set a temperature threshold x axis click Threshold the k icon then click and hold on the graph and drag the threshold line to the right This eliminates the threshold line for the lower temperatures Note This is useful when there is noise in the signal at low temperatures Reports This opens the Report Browser where a report can be chosen to preview A report can be generated based upon the currently selected channel or a multichannel genotyping report can be generated Results This displays the Melt Curve Results window which shows sample peaks Genotypes Click Genotypes and select the genotypes as shown below Genotype Abbrev Bin Bin B H omozygous M M ES RW IH eterozygous W wild Type OK Ca
79. al rotor holds tubes or Rotor Discs in the Rotor Gene Q It enables samples to spin in the instrument chamber and ensures that samples are correctly aligned with the optical system The rotor is secured with a Locking Ring Rotor Disc Rotor Discs are circular plates of vertically oriented reaction wells Rotor Disc formats for 72 and 100 reactions are available Rotor Discs are sealed using Rotor Disc Heat Sealing Film and the Rotor Disc Heat Sealer 13 2 Rotor Gene Q User Manual 11 2009 Appendix A Appendix A Technical data QIAGEN reserves the right to change specifications at any time Environmental conditions Operating conditions Power 100 240 V AC 50 60Hz 520 VA peak Power consumption 8 V standby Mains supply voltage fluctuations are not to exceed 1096 of the nominal supply voltages Fuse F5A 250 V fuse Heat dissipation Average 0 183 kW 632 BTU hour thermal load Peak 0 458 kW 1578 BTU hour Overvoltage Il category Air temperature 18 to 30 C 64 to 86 F Relative humidity 10 75 noncondensing Altitude Up to 2000 m 6500 ft Place of For indoor use only operation Pollution level 2 Environmental 3K2 IEC 60721 3 3 class Rotor Gene Q User Manual 11 2009 A 1 Appendix A Transportation conditions Air temperature 25 C to 60 C 13 F to 140 F in manufacturer s package Relative humidity Max 7596 noncondensing Environmental 2K2 IEC 60721 3 2 class Storage co
80. andard values are used to construct a standard curve to calculate unknown sample concentrations A calibrator is assigned a value of 1 and all other sample concentrations are calculated relative to this sample Rotor Gene Q User Manual 11 2009 7 73 Analysis User Interface Page This function allows the user to have different sample definitions and also separate experiments in the same run This is useful for analysis of different products in different channels Use the arrow buttons to move between the sample pages Use the New and Delete buttons to create and delete pages It is possible to have multiple sample definitions for the same channel in order to run multiple standard curves without multiplexing Simply define the samples of interest and their related standard curves on separate pages The single channel can then be analyzed with each set of definitions independently Sample pages can be labeled Page 1 Page 2 etc or they can be given any name e g Housekeeper This name will appear in reports When viewing the raw data the sample definitions used to display the data can be selected using the drop down menu next to the Options button T5 20 alt Scale Options Page 1 ind The sample page to use when performing an analysis can be selected in the Analysis window see Section 7 6 1 7 74 Rotor Gene Q User Manual 11 2009 Analysis User Interface x 2 Std Curve
81. ange the duration of the Hold click on the Hold Time mins and secs buttons H 9 New Open Save As Help The run will take approximately 83 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Cycling Insert before Remove Hold Temperature 95 deg Hold Time Ess mins Sp secs OK If performing Optical Denature Cycling a Hold can be used as a calibration step In this case a calibration melt is performed before the Hold By default this is configured for the first Hold in the run but may be changed if required Rotor Gene Q User Manual 11 2009 6 13 Operating Procedures Software Click on a cycle below to modify it Ins Cycling Inse Hold Temperature 95 deg Hold Time 3 mins 0 secs Calibration Step M Calibration Settings Acquiring to on tube 1 Ramp from 80 to 95 and hold for 3 mins 0 secs Offset 0 deg E dit For more information about Optical Denature Cycling see page 6 18 Cycling Cycling repeats the user defined temperature and time steps a specified number of times The number of repeats is set using the This cycle repeats X time s button A single cycle is displayed graphically as shown in the screenshot below Each step of the cycle can be altered The temperature can be changed by dragging the temperature line in the graph up or down The duration of th
82. angeschlossen ist Das ffnen oder Entfernen von Geh useteilen kann diese stromf hrenden Teile freilegen FR Risque d electrocution Toute interruption du conducteur de protection l int rieur ou l ext rieur de l instrument ou d connexion du raccord du conducteur de protection terre peut rendre l instrument dangereux ll est interdit d interrompre volontairement ce conducteur Pr sence de tensions mortelles dans l instrument Lorsque l instrument est reli au secteur les raccords peuvent tre sous tension et des parties sous tension peuvent tre d couvertes en ouvrant des capots ou en retirant des pi ces l exception de celles auxquelles il est possible d acc der manuellement WARNING A Electrical hazard w9 The instrument has an electrical compliance label which indicates the voltage and frequency of the power supply as well as fuse ratings The equipment should only be operated under these conditions Gef hrdung durch Elektrizit t Das Instrument besitzt ein Schild mit der Angabe des elektrischen Anwendungsbereichs das die Spannung und Frequenz des Netzteils und die erforderlichen Sicherungen vorgibt Das Ger t darf au erhalb der angegebenen Werte nicht benutzt werden Rotor Gene Q User Manual 11 2009 Safety Information FR WARNING A Risque d lectrocution L instrument porte une plaque d crivant le voltage la fr quence du bloc d alimentation lectrique
83. anscription PCR patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd to use it for the purchaser s own internal research No real time patent rights of any kind no right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel This product QuantiTect Primer Assays is compatible for use in the 5 nuclease process or the dsDNA binding dye processes covered by patents owned by Roche or owned by or licensed to Applera Corporation No license under these patents to practice the 5 nuclease process or the dsDNA binding dye processes are conveyed expressly or by implication to the purchaser by the purchase of this product RESEARCH USE ONLY A license under U S Patents 5 786 146 6 017 704 6 265 171 and 6 200 756 or their foreign counterparts is required to practice MSP Methylation Specific PCR The purchase price of this product EpiTect MethyLight PCR Kit includes limited nontransferable rights to use only this amount of the product to practice MSP solely for the internal research activities of the purchaser To obtain rights to practice MSP in any in vitro diagnostic application or to perform or offer commercial services of any kind using MSP contact OncoMethylome Sciences Certain specific embodiments of the process of multiplex PCR may be covered by patents of third parties in certain countries and may re
84. ard For help on an item hover Operator Rotor Gene Q Operator your mouse over the Notes Enter any notes for the particular HRM experiment Reaclion 25 Volume uL d Sample Layout 1 2 3 2 Skip Wizard Back Next gt gt Rotor Gene Q User Manual 11 2009 11 11 High Resolution Melt Analysis 4 Click on the Edit Profile button to modify the times and temperatures of the reaction New Run Wizard Temperature Profile This box displays help on elements in the wizard For help on an item hover your mouse over the item for help You can also click on a combo box to display help about its available settings Edit Profile Channel Setup Name Source Detector Gain Create New Green Yellow LER Orange Red Crimson Remove HRM 1 Reset Defaults Gain Optimisation Skip Wizard lt lt Back Next gt gt 5 Set an appropriate initial hold time This time depends on the type of DNA polymerase used The Type it HRM PCR Kit and the EpiTect HRM PCR Kit require a 5 minute activation time The default activation time is 10 minutes 11 12 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis x A U BH 9 New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Cycli
85. ase i 75 0 788 7 amp 0 76 8 mn 778 780 KE 79 73 5 0 as 810 p e Ann Melt phase Postmelt phase A typical HRM plot The melt curve plots the transition from the high fluorescence of the initial premelt phase through the decrease in fluorescence of the melt phase to the basal level of fluorescence at the postmelt phase Fluorescence decreases as DNA intercalating dye is released from dsDNA as it melts into single strands The midpoint of the melt phase at which the rate of change in fluorescence is greatest defines the melting temperature T of the DNA under analysis Before performing HRM analysis the target sequence must be amplified to a high copy number This is usually performed by PCR in the presence of a dsDNA intercalating fluorescent dye The dye does not interact with ssDNA but actively intercalates with dsDNA and fluoresces brightly when intercalated Change in fluorescence can be used to Rotor Gene Q User Manual 11 2009 11 1 High Resolution Melt Analysis measure the increase in DNA concentration during PCR and then to directly measure thermally induced DNA melting by HRM During HRM fluorescence is initially high because the sample starts as dsDNA Fluorescence decreases as the temperature is raised and DNA dissociates into single strands The observed melting behavior is characteristic of a particular DNA sample Using HRM the Rotor Gene Q can characterize samples based on sequence length GC content
86. astic cover Open the lid by sliding it towards the back A 72 Well Rotor this rotor is blue in color and 72 Well Rotor Locking Ring are inside Once you have unpacked the Rotor Gene Q proceed with installation Note Only connect the Rotor Gene Q to the computer with the USB and serial cables delivered with the instrument Do not use other cables Rotor Gene Q User Manual 11 2009 Installation Procedures 4 3 Accessories Rotor Discs and accessories can be ordered separately for use with the Rotor Gene Q For more details see Appendix C 4 4 Hardware installation Once the Rotor Gene Q has been unpacked proceed with installation as described below 1 Place the Rotor Gene Q on a level surface 2 Ensure that there is sufficient space behind the instrument for the lid to open fully 3 Ensure that the power switch at the back of the instrument can be reached easily 4 Do not obstruct the back of the instrument Ensure that the power cord can be easily detached if required to disconnect power to the instrument 5 Before switching on the Rotor Gene Q check the voltage selector switch at the back of the instrument The voltage selector switch is set by your sales distributor for the specific region Ensure that the setting is correct for the local mains voltage If it is not correct contact your sales distributor to arrange for the voltage selection to be changed WARNING Electrical hazard W10 A Voltage s
87. ave been used We recommend QIAGEN kits for HRM analysis Check if the reaction was set up correctly Check the cycling conditions Check the starting quality and quantity of the template We recommend QIAGEN kits for sample preparation Amplification or melt plots are saturated Gain set too high Use Auto Gain Optimisation see page 6 24 Confidence percentages have changed Normalization regions were moved by clicking and dragging Only move normalization regions if it is necessary to avoid parts of the melt curve Outliers are present in the data Inconsistent reaction setup Inhibitors present in sample Too little or degraded template Check if the correct reagents have been used Check that the tubes used are uniform Check that the same master mix was used for all samples Check the starting quality and quantity of the template Rotor Gene Q User Manual 11 2009 12 5 Troubleshooting This page intentionally left blank 12 6 Rotor Gene Q User Manual 11 2009 Glossary 13 Term Acquisition Bins Channel Gain Gain Optimisation Loading Block Locking Ring Glossary Description Acquisition is the collection of fluorescent data Each acquisition set of fluorescent data from a channel is displayed in the software as unanalyzed data in a Raw channel window This data can be analyzed using the options in the Analysis menu In a melt analysis bins are set to define
88. avec cet instrument peuvent amp tre dangereuses ou peuvent le devenir apr s que le protocole ait t effectu Toujours porter des lunettes de protection paire de gants et une blouse de laboratoire La personne responsable par exemple le Chef du laboratoire doit prendre les pr cautions n cessaires la s curit de l environnement du poste de travail et s assurer que les op rateurs de l instrument sont suffisamment form s et non expos s des quantit s dangereuses d agents infectieux comme d fini dans les fiches de donn es de s curit Material Safety Data Sheets MSDS ou des documents OSHA ACGIH ou COSHH L vacuation des vapeurs et d chets doit tre conforme tous r glements et dispositions l gales au plan national d partemental et local concernant la sant et la s curit Rotor Gene Q User Manual 11 2009 1 19 Safety Information WARNING AN Risk of fire W16 When cleaning the Rotor Gene Q with alcohol based disinfectant leave the Rotor Gene Q lid open to allow flammable vapors to disperse Only clean the Rotor Gene Q when worktable components have cooled down Feuergefahr Beim Reinigen des Rotor Gene Q mit einem auf Alkohol basierenden Desinfektionsmittel muss die Haube des Rotor Gene Qs offen gelassen werden damit die brennbaren D mpfe entweichen k nnen Den Rotor Gene Q nur reinigen sobald die entsprechenden Module auf der Arbeitsfl che abgek hlt sind Ri
89. ays wear safety glasses 2 pairs of gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are suitably trained and not exposed to hazardous levels of infectious agents as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws Rotor Gene Q User Manual 11 2009 Safety Information 1 5 Chemicals WARNING AN Hazardous chemicals W15 Some chemicals used with this instrument may be hazardous or may become hazardous after completion of the protocol run Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America ACGIH American Conference o
90. cates that this product must not be disposed of with other waste it must be taken to an approved treatment facility or to a designated collection point for recycling according to local legislation The separate collection and recycling of waste electronic equipment at the time of disposal helps to conserve natural resources and ensures that the product is recycled in a manner that protects human health and the environment Rotor Gene Q User Manual 11 2009 A 3 Appendix A QIAGEN accepts its responsibility in accordance with the specific WEEE recycling requirements and where a replacement product is being supplied by QIAGEN provides free recycling of its WEEE marked electronic equipment in Europe If a replacement product is not being purchased from QIAGEN recycling can be provided upon request at additional cost To recycle electronic equipment contact your local QIAGEN sales office for the required return form Once the form is submitted you will be contacted by QIAGEN either to request follow up information for scheduling collection of the electronic waste or to provide you with an individual quote Rotor Gene Q User Manual 11 2009 Appendix B Appendix B This appendix describes the mathematical techniques used in more detail Quantitation Calculated concentrations are obtained from a simple linear regression model with the known values the log concentrations x and the experimental values the C values y The
91. cation The run is given a file name that consists of the template used and the date of the run A serial number 1 2 etc is also included in the file name to allow automatic naming of numerous runs that use the same template on the same day EC 21x soarce emer E Three Step with Melt 2008 12 04 1 x Save as type Run File rex D Cancel 6 6 Rotor Gene Q User Manual 11 2009 Operating Procedures Software 6 1 4 6 2 Sample setup Once the run has started the Edit Samples window allows samples to be defined and described BEE File Edit Format Security Standard Rotor Style m Settings Given Cone Format 123 457 zl Unit Esel v More Options Samples E gt Edit Reset Default Gradient LY ed E CR ES ss I C Ip Name Type Groups Given Conc Selected A1 R1000 Standard 1 000 Yes A2 R1000 Standard 1 000 Yes H A3 R100 Standard 100 Yes 44 R100 Standard 100 Yes 45 R10 Standard 10 Yes Ap R10 Standard 10 Yes A Ri Standard 1 Yes A8 R1 Standard 1 Yes ES pi RNTC NTC Yes M B2 RNTC NTC Yes B3 11000 Standard 1 000 Yes B4 11000 Standard 1 000 Yes r Page Name Paget med 2 New A Synchronize pages Undo DK Cancel Help The Edit Samples window appears after the run has started so that the user can use this time to enter sample names For information about setting up sample definitions in the Edit Samples window
92. cautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws Rotor Gene Q User Manual 11 2009 Safety Information DE FR Gef hrliche Chemikalien Einige der in Verbindung mit diesem Ger t verwendeten Chemikalien sind gesundheitsgef hrdend oder k nnen nach Beendigung eines Protokoll Durchlaufes gesundheits gef hrdend werden Es sollten immer Sicherheitsbrille Handschuhe und ein Laborkittel getragen werden Der Betreiber der Anlage ist f r die Gew hrleistung der Sicherheit am Arbeitsplatz verantwortlich Er hat sicherzustellen dass die Bediener des Ger tes ausreichend geschult sind und nicht gesundheitsgef hrdenden Konzentrationen toxischer Substanzen chemischer oder biologischer ausgesetzt sind so wie dies in den Sicherheitsdatenbl ttern oder in anderen zu beachtenden Dokumenten festgelegt ist Bei der Behandlung von Abluft und bei der Abfallbeseitigung sind alle gesetzlichen Regelungen zur Gesundheit und Sicherheit auf nationaler regionaler und lokaler Ebene zu ber cksichtigen Substances chimiques dangereuses Certaines substances chimiques utilisees
93. ccessories M Liability clause Rotor Gene Q User Manual 11 2009 2 1 Introduction 2 2 General Information 2 2 1 Technical assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Services Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding the Rotor Gene Q or QIAGEN products in general do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information call one of the QIAGEN Technical Services Departments or local distributors see back cover For up to date information about the Rotor Gene Q visit www giagen com goto Rotor GeneQ 2 2 2 Policy statement It is the policy of QIAGEN to improve products as new techniques and components become available QIAGEN reserves the right to change specifications at any time In an effort to produce useful and appropriate documentation we appreciate your comments on this user manual Please contact QIAGEN Technical Services 2 2
94. ch run Analysis Concentration analysis operates by relating the fluorescence level to a concentration value Two analysis models are available The optimal analysis to chose depends on the chemistry and application Linear Regression analyzes data by assuming a linear relationship and estimating unknown values on the basis of a generated linear model It determines measurement error by examining the deviation of the readings from a linear model 7 60 Rotor Gene Q User Manual 11 2009 Analysis User Interface If concentration readings are linear this is the most suitable analysis because it provides statistical analysis of variation ANOVA to the user Spline Curve Fit assumes only that concentration values increase with fluorescence While this approach makes estimates of nonlinear data more accurate it cannot provide ANOVA as it does not assume a linear model 7 6 11 High Resolution Melt analysis High resolution melt HRM analysis characterizes samples based on sequence length GC content and complementarity HRM analysis is used in genotyping applications such as analysis of gene mutations or single nucleotide polymorphisms SNPs and in epigenetics applications for analysis of DNA methylation status HRM analysis provides accurate results and savings on probe and label costs compared to other methods To perform the analysis select Other and then High Resolution Melt Analysis in the Analysis window
95. changing colors of curves adding annotations changing fonts and adjusting data points amp TeeChart Office test tee OI x Ele Edk View Tools Heb Os a le QO gt KB ra mxe r gi 2207 amesa Galt Ka i kr V 2 Heterozygous bat 3 Widtype ks v 4 Heterozygous kr g 5 Heterozygous iw 8 4 Rotor Gene Q User Manual 11 2009 8 5 Additional Functions Data export To export data in various formats select Data tab in the Export Dialog window The exported file contains the raw data points used in the graph Export Dialog Fi Picture Native Data l Include Series all 5 Point Index Eormat v Point Labels Ted K Heade C XML Point Colors C HTML Table Delimiter Excel T Exporting raw data and analysis data can also be performed by selecting Save As under the File menu see Section 7 5 Spanner wrench icon The spanner wrench icon 2 appears at the bottom left of the main window Clicking on the spanner wrench icon enables several options These options can also be accessed by right clicking on the graph Rotor Gene Q User Manual 11 2009 8 5 Additional Functions Adjust Scale Autoscale Revert to Default Scale Export Copy Chart to Clipboard Edit Chart in TeeChart Office Print Digital Filter Select All Named Samples Show Pinpointer Grouping
96. commend preparing a test sample containing all the reaction components Place the test sample in the Rotor Gene Q and use Gain Optimisation to determine the best gain setting If the gain chosen by Gain Optimisation results in a poor signal then the Target Sample Range should be increased If it results in a signal that is saturated then the Target Sample Range should be decreased To perform Gain Optimisation click on the Gain Optimisation button in New Run Wizard window 3 see Section 6 2 3 6 24 Rotor Gene Q User Manual 11 2009 Operating Procedures Software New Run Wizard Temperature Profile This box displays help on elements in the wizard For help on an item hover your mouse over the item for help Y ou can also click on a combo box to display help about its available settings Edit Profile Channel Setup ee as Detector Gan 4 Create New Green 470nm 510nm Pg Yellow Orange Edit Gain Red Crimson HRM Gain Optimisation Skip Wizard lt lt Back Next gt gt Remove Reset Defaults The Auto Gain Optimisation Setup window appears This window enables optimization by automatically adjusting the gain settings until the readings for all selected channels fall within or below a certain threshold Rotor Gene Q User Manual 11 2009 6 25 Operating Procedures Software Auto Gain Optimisation Setup x Optimisation T
97. con has doubled in every cycle In the raw data the signal should be doubling in the exponential phase For example if the signal was 50 fluorescence units at cycle 12 and then 51 fluorescence units at cycle 13 it should increase 7 42 Rotor Gene Q User Manual 11 2009 Analysis User Interface to 53 fluorescence units at cycle 14 All of the amplification values for each sample are averaged to produce the amplification value that is shown on the right of the screen under the toggler The greater the variation between the estimated amplification values of each sample the larger the confidence interval will be indicated by the value after the sign The confidence interval for a large sample number N gives a 68 396 probability that the true amplification of the samples lies within this range 1 standard deviation By doubling the interval a 95 496 confidence interval for a large N is achieved Calibrator Replicate As in the delta delta C method a calibrator sample is required and measurements are relative to this calibrator sample Replicates of the calibrator can be analyzed since if multiple sample positions have the same name the average of the takeoff points of these samples will be used To use this feature correctly ensure that replicates have identical names Results Average Amplification 1 78 0 04 The average amplification is used to calculate expression For example a sample with a low amplification
98. dard curve for each gene The concentration for each gene is quantified according to its standard curve The expression of the gene of interest is then normalized with the normalizing gene often a housekeeping gene It is important that the standards and replicate samples are designated correctly during sample setup see Section 6 1 4 In particular corresponding samples must have the same name in each analysis In a multiplex reaction where the tube positions of the gene of interest and the normalizing gene are the same one set of sample definitions is sufficient If performing relative analysis with a normalizing gene using a single channel i e reactions are run in separate tubes using the same fluorophore then 2 sample pages should be created The first should label the tube positions with sample names for the gene of interest with the other positions left unnamed The second should label the positions used for the normalizing gene The software will then match samples across the 2 analyses based on their names Expression analysis using the two standard curve method Data can first be analyzed for each gene using quantitation analysis Otherwise the results for each gene will 7 30 Rotor Gene Q User Manual 11 2009 Analysis User Interface automatically be determined using the Autofind Threshold tool 1 From the Analysis window select the 2 Std Curve Rel tab Click New Analysis Quantitation Mel
99. e The software must be installed with the Force Authentication through Windows NT Logon option Note The Security menu will not appear if you are logged into a Linux Samba domain You must have either a local logon or a Windows server to use the security features Rotor Gene Q User Manual 11 2009 7 81 Analysis User Interface 7 9 1 Configuration This section describes how to set up the system to run Rotor Gene Q software securely To use the security features the software must be installed with the Force Authentication through Windows NT Logon option This queries the Windows domain for your access level and credentials and is essential for providing the accountability and security features Running as administrator Many users run their computers as an administrator with no password While this is convenient it makes it impossible to determine who is using the computer This eliminates accountability and prevents many Rotor Gene Q software security measures from activating When running as an administrator all the software features are enabled Therefore running as an administrator ensures that users who do not need security features can access all the software features Creating a new user account Create user accounts for each user of the software For each user repeat the steps below until all accounts have been created 1 To create a new user select Start Settings Control Panel 2 Double c
100. e a single tube position It is not currently possible to analyze a setup in which a sample position is a positive control for one channel and a negative control for another Although only one sample definition per tube position is given in the Edit Samples window normalization occurs independently for each channel If a tube position is a positive control for at least one channel it should be specified as a positive control in the Type column of the Edit Samples window Otherwise its type should be Sample This also applies for negative controls For example if a sample is a positive control in the green channel but not in the yellow channel the sample should 7 56 Rotor Gene Q User Manual 11 2009 Analysis User Interface still be defined as a positive control Since the highest positive control in each channel is used if there is least one positive control in the yellow channel that amplifies the definition of the sample as a control for the green channel is ignored Threshold The threshold is used to determine the percentage expression required for a reaction in each channel Once the positive and negative controls have been defined all channels will be normalized to the same 0 100 scale For this reason only one threshold is needed even when analyzing multiple channels Click and drag the threshold line to an area between O and 100 The threshold should not be too close to samples on
101. e change for heterozygotes Difference plots are an aid to visual interpretation They plot the difference in fluorescence of a sample to a selected control at each temperature transition Difference plots provide an alternative view of the differences between melt curve transitions Note First derivative melt curve analysis as used by the standard Melt option in the Analysis window is considered inappropriate for HRM analysis This is because any derivation of the data adds artificial noise and makes data interpretation more difficult The following steps describe the analysis of HRM results using Rotor Gene Q software 1 Select the HRM option from the Analysis window x 2Std Curves Rel Other Quantitation Melt w HRM A HRM Page 1 Rotor Gene Q User Manual 11 2009 11 19 High Resolution Melt Analysis 2 BERotor Gene Q Series Software high resolution melt demo 18 x File Analysis Run Gain View Window Help Windows appear showing the raw data the normalized graph and the results The raw data window allows adjustment of the regions of normalization Normalization allows all the curves to be compared with the same starting and ending fluorescent signal level to aid interpretation and analysis Two cursors per region are provided defaulted to the ends of the curve The data points within the regions are used to normalize fluorescence the y axis only for the start Region 1 and end Re
102. e containing a Denature step and an Optical Denature Cycle step will appear The ramped region at the beginning of the run represents the calibration process The green dots represent the acquisitions taken each cycle during heating The blue dots represent the acquisition at the end of the anneal step at 60 C Note that while the profile shows each step with the same denature temperature this may not be the case If the sample requires slightly longer to melt towards the end of the run the optical denature process waits for the melt according to the fluorescent data and not according to time For this reason the temperature trace may vary for each cycle 2 Click on the first half of the graph with the Optical Denature symbol 3 The Calibration Settings information appears on the left of the screen This cycle repeats 45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle Optical Denature Calibration Settings Acquiting to RealTime on tube 1 Ramp from 80 to 95 land hold for 3 mins 0 secs Offset 0 deg Edit 60 deg for 40 secs OK 3 The Calibration Settings information is usually correct To modify it if necessary click Edit The Calibration Settings window appears Rotor Gene Q User Manual 11 2009 6 21 Operating Procedures Software Calibration Settings x Settings The Optical Denature Calibration setti
103. e couvercle et l appareil Moving parts W19 In case of breakdown caused by power failure remove the power cord and wait 10 minutes before attempting to manually open the lid Bewegliche Ger teteile Bei einem durch Stromausfall entstandenen Ausfall des Ger tes das Stromkabel entfernen und 10 Minuten warten bevor der Zentrifugendeckel manuell ge ffnet werden kann El ments mobiles Dans le cas d un arr t caus par une panne de courant retirer le c ble d alimentation et attendre 10 minutes avant d essayer d ouvrir manuellement le couvercle de la machine WARNING AN Risk of overheating W20 To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the Rotor Gene Q Slits and openings that ensure the ventilation of the Rotor Gene Q must not be covered Rotor Gene Q User Manual 11 2009 1 21 Safety Information berhitzung des Ger tes Zur Sicherstellung einer ausreichenden Bel ftung des Rotor Gene Q muss ein Mindestabstand von 10 cm an den Seiten und an der R ckseite des Ger tes eingehalten werden L ftungsschlitze und ffnungen des Ger tes nicht abdecken Risque de surchauffe Laisser un espace d au moins 10 cm sur les c tes et l arri re du Rotor Gene Q pour assurer une ventilation efficace Les grilles et prises d air assurant la ventilation du Rotor Gene Q ne doivent pas tre couvertes WARNING Hot surface
104. e no template controls select them in the sample type column then press N for NTC All samples will be converted to NTC A complete sample description can be saved as a sample file smp and loaded into future runs with the same sample configuration This tab in the Edit Samples window provides an alternative way of entering sample names Select replicates by clicking and dragging the mouse pointer over the rotor image The list on the right of the window will update The sample name can be typed in and this will set the same name for the current selection The software recognizes these wells as replicates 7 76 Rotor Gene Q User Manual 11 2009 Analysis User Interface BER File Edk Format Security Standard Rotor Style D 9 d e a F Label Wells As 1 2 3 Mss e eo ree EE ee The Rotor Style tab provides a cutdown version of the Standard tab and is designed for users who want to setup sample names and colors quickly It is not possible to define some settings such as the whether the sample represents a standard or the known concentration of each standard in this tab If these need to be defined the standard tab should be used Sample Page Suitability To access the Sample Page Suitability window click on More Options in the Edit Samples window and then click on Define Suitabilities The Sample Page Suitability window allows users to match sample pages to channels For examp
105. e step can be changed by dragging the temperature boundary in the graph left or right Alternatively click on the step and use the temperature and time buttons to the left of the graph Rotor Gene Q User Manual 11 2009 Operating Procedures Software Steps can be added or removed from the cycle using the and buttons at the top right of the graph x E 9 New Open Save As Help The run will take approximately 89 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after Insert before Remove This cycle repeats 45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle 95 deg for 20 secs 20 seconds Not Acquiring Sa 72 deg for 20 secs IT Long Range 60 deg for 20 secs IT Touchdown Long Range Checking this box increases the hold time of the selected step by one second with each new cycle Touchdown Checking this box decreases the temperature by a specified number of degrees for a specified number of initial cycles This is then shown in the display Acquisition Data can be acquired on any channel at any cycling step To set a channel to acquire data click on the Not Acquiring button if a channel has already been set to acquire at this step then the acquiring channels are listed here Rotor Gene Q User Manual 11 2009 6 15 Operatin
106. e the name and the save location This is the default format Template This saves the profile setup and associated settings but not the run data The template can be used to initiate future runs Rotor Gene Q User Manual 11 2009 7 7 Analysis User Interface Run Archive LIMS Export Excel Data Sheet Excel Analysis Sheet LinReg Export Format Matlab Export This saves in a more compact file format Save files in this format before they are e mailed This reduces the time required to send the file and ensures that files are not corrupted by e mail clients This saves the analysis in LIMS compatible formats according to the requirements of the user Please contact QIAGEN Technical Services for more information This exports all the raw channels to an Excel sheet Only the selected samples are exported This exports all the analysis in the current run into a single Excel sheet This exports all raw channel data into a format that can be read by LinReg an efficiency analysis tool See Exporting To LinReg below for more details This exports the data into a format that can be read by the scientific package Matlab or its open source equivalent Octave This may be useful for methods research Exporting To LinReg LinReg is a tool developed by C Ramakers and coworkers The LinReg tool is available on request e mail bioinfo amc uva nl subject LinRegPCR Rotor Gene
107. ecome corrupt This may happen if the software computer is shut down without exiting correctly for example during a power outage Delete the file C Program Files Rotor Gene 6000 Software Templates normal ret and then restart the software Rotor Gene Q User Manual 11 2009 12 3 Troubleshooting Comments and suggestions Can open the serial This error occurs on software startup if the port COMx software cannot communicate with the instrument via the configured COM port This is commonly caused by faulty cables loose cables faulty serial ports faulty USB ports a USB driver problem or a USB to serial converter driver problem Reconnect or replace the cable Reinstall the appropriate drivers Start the software in Virtual Mode and select Setup Auto Detect button from the File menu to reset the configured COM port 12 4 HRM troubleshooting Comments and suggestions Unable to run HRM Rotor Gene Q model is Contact your local QIAGEN representative not HRM equipped No HRM data obtained Incorrect setup Check filter settings Check if the rotor type is correct Check if the correct reagents have been used Check if the reaction was set up correctly Run a positive control experiment i e an assay that is known to provide results 12 4 Rotor Gene Q User Manual 11 2009 Plots look jagged Poor or no amplification Troubleshooting Comments and suggestions Check if the correct protocols and reagents h
108. ed its windows are arranged to fit in with those already on the screen If many windows are displayed this can be cumbersome Close the windows you do not require then click Arrange on the toolbar The windows are automatically arranged according to the Smart Tiling method Alternatively select another arrangement method by clicking the arrow next to the Arrange button Clicking the right mouse button on the name of an analysis also provides additional options Quantitation Melt Cycling A Green Page 1 Cycling Orange Shen Cycling A Rec Hde Bamnve amp nalusiz Cycling AY ellow Show This displays the selected analysis Hide This hides the selected analysis Remove This removes the selected analysis Analysis completely This means that any normalization settings or melt bins set up in the analysis will be lost Quantitation Select the Quantitation tab in the Analysis window and then double click on the channel name or select the channel and then press the Show button to open the channel of interest Three windows appear the main screen the standard curve and the results Rotor Gene Q User Manual 11 2009 Analysis User Interface Reports Reports Reports opens the Report Browser window where a report of the current analysis can be generated There are 3 options standard report full report and concise report Double click on the desired option to open the report in the P
109. egional Options Click on the Numbers tab Os rn cet ope ced Rotor Gene Q User Manual 11 2009 12 1 Troubleshooting 7 Note down what is used as the decimal symbol and what is used as the digit grouping symbol On a German system for example the comma will be used for decimals and the period is used as a digit grouping symbol Click on the Currency tab Change the decimal symbol and the digit grouping symbol to be the same as in the Numbers tab 10 Click OK 11 Rerun the Rotor Gene Q software 12 2 Rotor Gene Q User Manual 11 2009 Troubleshooting 12 3 Rotor Gene Q troubleshooting Comments and suggestions General instrument errors Machine unplugged serial timeout error Object variable or with block variable not set This error occurs if the instrument does not communicate with the software after a defined timeout interval It is often caused by an instrument fault or by excessive activity from the PC which causes a packet to be lost Common software related causes include processor intensive tasks such as antivirus resident protection or antivirus scheduled scans wireless cards or infrared cards Disable or uninstall the relevant processor intensive software task Common instrument related causes include faulty USB serial cables or loose USB serial cables Reconnect or replace the cable This error occurs on software startup if the default experiment template file has b
110. election may only be changed by your sales distributor or authorized personnel Users should not attempt to change voltage selection Doing so may void warranty WARNING A Electrical hazard w11 Do not change the voltage selector switch setting while the instrument is connected to the mains Otherwise you may cause damage to the instrument and or blow the fuse Rotor Gene Q User Manual 11 2009 4 3 Installation Procedures 6 Connect the USB cable or RS 232 serial cable supplied to a USB or communications port on the back of the computer 7 Connect the USB or RS 232 serial cable to the back of the Rotor Gene Q 8 Then connect the Rotor Gene Q to the power supply Connect one end of the AC power cord to the socket located at the rear of the Rotor Gene Q and the other end to the AC power outlet nmm On off switch Power supply port Serial number Cooling fan Serial port USB port 4 5 Software installation 1 To install the Rotor Gene Q software insert the installation CD into the CD drive of the computer 2 Select Install Operating Software in the window which appears Follow the setup wizard for easy installation 4 4 Rotor Gene Q User Manual 11 2009 Installation Procedures Rotor Gene Q Pure Detection Install Operating Software go Exit 3 Once the software has been installed switch on the Rotor Gene Q by moving the switch located at the back on the right hand s
111. ent 7 58 O Operation conditions 1 5 A 1 hardware 5 1 software 6 1 Optical denature cycling 6 18 Optical system 3 3 Optical temperature verification 10 1 Outlier removal 7 26 P Page 7 3 7 5 7 74 Perform last run 6 2 6 8 Port 4 6 7 10 Profile progress 7 69 Q Quantitation 7 12 Quantitation results window 7 20 Quenched FRET 6 2 Quick start wizard 6 1 R Raw channels 7 1 Reaction setup 5 4 Report browser window 7 9 7 13 7 40 Rotor 36 Well 5 1 72 Well 5 2 Rotor Disc 100 5 2 Rotor Disc 72 5 2 selection 6 4 6 9 specifications 5 3 types 5 1 Rotor Disc heat sealing 5 8 setup 5 8 Rotor Disc 100 5 2 Rotor Disc 72 5 2 Rotor Disc OTV Kit 10 1 Index 2 Rotor Gene Q User Manual 11 2009 Index Run new 7 6 open 7 7 pause 7 63 save 7 7 settings 7 63 signatures 7 94 start 7 62 stop 7 63 S Safety biological 1 5 chemicals 1 7 electrical 1 3 heat hazard 1 9 mechanical hazards 1 8 proper use 1 2 waste disposal 1 7 Sample page suitability window 7 77 Sample types 7 72 Scaling 8 1 Scatter graph analysis 7 46 Security 7 67 7 81 Serial number 4 6 Setup window 7 9 Slope 7 28 Software updates 4 8 version 4 7 Spanner icon 8 5 Specifications hardware A 2 optical A 3 thermal A 2 Standard curve 7 13 calculation 7 16 export 7 15 formula 7 15 7 29 import 7 16 overlay 7 15 two standard curve method 7 30 Storage A 2 Su
112. f Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom WARNING AN 1 6 Risk of fire W16 When cleaning the Rotor Gene Q with alcohol based disinfectant leave the Rotor Gene Q door open to allow flammable vapors to disperse Only clean the Rotor Gene Q when worktable components have cooled down Toxic fumes If working with volatile solvents or toxic substances you must provide an efficient laboratory ventilation system to remove vapors that may be produced Waste disposal Used consumables and plasticware may contain hazardous chemicals or infectious agents Such wastes must be collected Rotor Gene Q User Manual 11 2009 1 7 Safety Information and disposed of properly according to local safety regulations 1 7 Mechanical hazards The lid of the Rotor Gene Q must remain closed during operation of the instrument WARNING Moving parts W17 N To avoid contact with moving parts during operation of the Rotor Gene Q the instrument must be operated with the lid closed WARNING Risk of personal injury and material damage W18 N Open and close the lid of the Rotor Gene Q carefully to avoid trapping fingers or clothing Rotor Make sure that the rotor and locking ring are installed correctly If the rotor or locking ring show signs of mechanical damage or corrosion do not use the Rotor Gene Q contact QIAGEN
113. f run information run filename analysis date operator and any associated notes Rotor Gene Q User Manual 11 2009 7 63 Analysis User Interface The window contains all information except for the profile required to configure a run After a run has finished the following information is displayed in this window cycler used gain settings number of channels and time of start and finish Genera Machins Cptione Messages Channels Tubs Layout Security Filenarre F Departmenls QC E xperimentalD ata R 3 3K F G60004F1 2052740 ark Operator Notes Reaction Volume O yL hiseters to the total volume ol the mature in the tube Other Run Lhannels saved tor Ihis rur Informaton Cycling A Green Cycling D Yellow Cycling C Orange Cycling D Bed Created with template C Documents and SettingsNlabpc CORBETT Desktop T 2mp RG600C Hun has tinished Started at 15 12 2005 9 38 58 AM Finished at 16 12 2005 9 52 40 AM Offset Coefficient 0 Progiam Version 3otor Gene 6000 Series Software 1 7 Build 3 Machine Serial Na 000627 Machine Options This tab displays settings for the configuration of the Rotor Gene Q 7 64 Rotor Gene Q User Manual 11 2009 Analysis User Interface The rotor should be set to that currently installed in the Rotor Gene Q If opening an existing run this setting will reflect the rotor that was installed in the cycler at that time Messages
114. ficient reaction took place during the exponential phase Negative percentages indicate that during the exponential phase the fluorescent signal declined Current research is not conclusive on the precise levels of efficiency needed to distinguish genuine reactions from contamination and other effects For this reason we recommend using this feature conservatively with the assumption that any sample with a genuine reaction will have some visible exponential phase with some increase in fluorescence Setting this value higher than 096 will exclude some samples with inefficient but perceptible increase in fluorescence whereas setting below 096 will display samples that Rotor Gene Q User Manual 11 2009 7 27 Analysis User Interface 7 28 decreased in fluorescence during the exponential phase which should clearly be excluded Note If a value is excluded due to activation of either of these techniques the C value in the Quantitation Results window will be labeled to indicate the exclusion In the image below samples 19 20 and 22 were excluded due to the Reaction Efficiency Threshold i Given Conc Cop 19 Jeck Unknown NEG R Eff 20 3e Unknown NEG R Eff 22 4a Standard NEG R Eff 50 23 4b Standard 33 03 50 Slope amplification reaction efficiency The slope M of a reaction shown in the Standard Curve window can be used to determine the exponential amplification and efficiency of a reaction usi
115. forme du Rotor Gene Q peut causer des blessures ou des d t riorations de l instrument Le Rotor Gene Q ne doit tre utilis que par du personnel qualifi qui a t form de facon appropri e Seul un ing nieur du service apr s vente QIAGEN est autoris effectuer des travaux d entretien sur le Rotor Gene Q WARNING Risk of personal injury and material damage W2 Rotor Gene Q is a heavy instrument To avoid personal injury or damage to the instrument take care when lifting Rotor Gene Q User Manual 11 2009 Safety Information DE Verletzungsgefahr und Besch digung des Ger tes Der Rotor Gene ist ein schweres Instrument Bitte mit Vorsicht heben um Verletzungen oder einen Schaden am Ger t zu vermeiden FR Risque de dommages corporels et mat riels Le Rotor Gene Q est un instrument lourd Pour viter de se blesser ou d endommager la machine soulever avec precaution WARNING Risk of personal injury and material damage W3 Do not attempt to move the Rotor Gene Q during A operation DE Verletzungsgefahr und Besch digung des Ger tes Den Rotor Gene Q w hrend eines Laufes nicht bewegen FR Risque de dommages corporels et mat riels Ne pas essayer de bouger le Rotor Gene Q pendant son fonctionnement WARNING Risk of personal injury and material damage w4 A DE Do not try to open the lid during an experiment or while the Rotor Gene Q is spinning Otherwise if you overc
116. g Procedures Software 10 seconds Not Acquiring Long Range Touchdown After clicking the Not Acquiring button the Acquisition window appears Same as Previous New Acquisition m r Acquisition Configuration Available Channels Acquiring Channels gt reen Crimson Yellow To acquire from a channel select it from the list in the left and click gt To stop acquiring from a channel select it in the right hand list and click lt To remove all acquisitions click lt lt Dye Chart gt gt Dye Channel Selection Chart D I Quasar 670 Alexa Fluor 533 rm 660nm Crimson Quasar705 Alexa Fluor 680 RM 4 amp 0nm 510nm SYTO 9 EvaGreen To set a channel to acquire select the channel and move it from the Available Channels list to the Acquiring Channels list using the gt button To remove a selected 6 16 Rotor Gene Q User Manual 11 2009 Operating Procedures Software channel from the Acquiring Channels list use the button The l button removes all the channels from the Acquiring Channels list Clicking the Don t Acquire button also removes all acquisitions from the step If more than one cycling sequence is included in the profile the acquired data can be appended to the data acquired from the earlier cycling Use the Same as Previous drop down menu to select the cycling step to which the data should be appended
117. ge Stat e83 End ar Confidence Percentage Theshoa 30 x Rotor Gene Q Series Software 1 7 Build 94 4 Click on the Genotypes button to define the genotypes Input each genotype category name and select a representative sample for each from the sample list zii xl mutation wild type heterozygote DK Cancel Rotor Gene Q User Manual 11 2009 11 21 High Resolution Melt Analysis 11 22 its Select a Control x Please select the control that is representative of this genotype Currently Selected 198 2 197 3 negl 41196 5 195 6 neg2 7 202 8 201 fo 200 E Select Select None Cancel 5 View the difference plot by selecting the Difference Graph tab Then select the genotype you wish to compare all other samples against using the drop down menu at the top of the window In the example shown all samples are plotted subtracted from an average plot of all samples labeled Mutation 1 Rotor Gene Q Series Softwar e high resolution melt demo HRM Normalised Graph Melt A from 82 FarRed Page 1 laj xj X File Analysis Run Gain View Window Help 81 sl 7 5 S 3 27 a A A Uh UM oj s B8 WD HG N i New Open Save Start Pause Stop Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Melt A FarRed Melt A from 82 FarRed No
118. gion 2 of the melt plot Data outside the set regions are ignored Adjust the regions to encompass representative baseline data for the premelt and postmelt phases Widening the regions by clicking and dragging allows the software to adjust for the slope of the baseline To ensure curves normalize effectively avoid widening the normalization regions into the melt phase bd New Open Save Start Pau Channels Melt A FarRed Mel A from 62 FerRed view F ra Min Vy Si m S Settings Progress Profile Temp Samples Analysis Reports Arrange H E 9 Help Stop Fluorescence E HRM Analysis Melt A from 82 FarRed Page 1 Dj x Page Page 1 Reports Resuks P D 825 83 0 835 84 0 845 85 0 855 E Edit Samples tH a Normalised Graph Genotypes NamedOn Ain All Off Adjust Scale Auto Scale Normalisation Regions gt Leading Range EI HRM Normalised Graph Melt A fr 0 x ime HRM Results Melt A from Sp Stat 24 Normalised Graph Difference Graph No C Name Genotype Confidence End 28 H T 1 198 mutation 100 00 100 2 197 heterozygote 100 00 Trailing Range 8 4 Wis mutation 95 72 tb 3 5 195 heterozygote 97 58 z 50 7 Ni heterozygote 97 80 End 897 H E NG
119. h is used as a positive control to call unknown samples automatically 101 xl Control mutation wild type heterozygote For more details on HRM analysis see Section 11 Run menu Start Run This option starts the defined temperature profile with the current gain settings Before the run starts the Profile Run 7 62 Rotor Gene Q User Manual 11 2009 7 7 2 Analysis User Interface Confirmation window appears A graphical representation of the temperature profile is displayed along with the gain settings for each channel Pause Run This option enables a run to be paused and resumed Pausing and resuming can seriously affect the results of a run For this reason a marker in the data will indicate that the run was paused and the length of the pause A message is also placed in the messages tab of the Run Settings window see page 7 65 You can pause the run on this screen marker will be recorded in the channel data to show the duration of the pause Resume Help Close WARNING A Hot surface W22 When pausing a run the Rotor Gene Q will not be cooled completely to room temperature Exercise caution before handling the rotor or any tubes in the instrument 7 7 3 7 8 7 8 1 Stop Run If this option is selected a prompt will appear asking for confirmation that the run should be stopped View menu Run Settings General This window allows the setup o
120. h user Access is divided into the following areas Rotor Gene Q Operator can perform runs but cannot generate reports or perform analysis Rotor Gene Q Analyst can analyze run data and generate reports but cannot perform new runs Rotor Gene Q Operator and Analyst has the capabilities of both roles Administrator can unlock sample names and perform all operations of Analysts and Operators None access to the software is denied 7 86 Rotor Gene Q User Manual 11 2009 Analysis User Interface To assign roles 1 Log in to Windows as an administrator or use the Rotor Gene Q Software Login icon to open the software and log in x Run Rotor Gene As Other User Domain CORIT198 Username linsonay Cancel Password 2 Once the software is open click on the Security menu The first time the Security menu is accessed Rotor Gene Q software configures a number of system groups that will control access to the software Rotor Gene Q Series Software x K RG Groups do not exist on the domain Would you like them to be created 9 e 3 Click Yes The User Admin window appears In the top panel all the users of the computer are displayed Some accounts are used by the system and so will be unfamiliar The bottom pane shows the groups assigned to the user Rotor Gene Q User Manual 11 2009 7 87 Analysis User Interface LIINC E Current User linsongy
121. he Quick Start wizard or the Advanced wizard which appear when the software is started up The Quick Start wizard is designed to allow the user to start the run as rapidly as possible The Advanced wizard enables more options such as configuration of Gain Optimisation and volume settings For convenience the wizards have a number of templates with default cycling conditions and acquisition channels To change the wizard type select the appropriate tab at the top of the New Run window Quick Start wizard The Quick Start wizard allows the user to start the run as rapidly as possible The user can select from a set of commonly used templates and enter the minimum of parameters to get started The Quick Start wizard assumes that the reaction volume is 25 ul For other reaction volumes use the Advanced wizard see Section 6 2 As a first step select the desired template for the run by double clicking on the template from the list in the New Run window Rotor Gene Q User Manual 11 2009 6 1 Operating Procedures Software x Buick Start Advanced Imports the cyding and acquisition and sample definitions from the last run open in the software erform Last Run Three Step with Melt ee Two Step ee 1S Quenched FRET e d e Nucleic Acid Concentration Measurement i Other Runs Open A Template In Another Folder v Show This Screen When Software Opens
122. he display Samples with a brightly colored cell are displayed while samples with a faded cell are not displayed Samples can be switched on or off by clicking on the cell or by dragging the mouse pointer across several cells at time The Bank On and Bank Off buttons hide or display respectively all samples currently visible in the list The scroll bar can be used to display the next group of samples Note The number of displayed samples is dynamic and depends on the space available in the window Rotor Gene Q User Manual 11 2009 7 3 Analysis User Interface Clicking Named On shows only those samples that have been given a name This is a quick way to show only relevant samples Clicking All On or All Off displays all or none of the samples in the rotor respectively Pressing the Edit Samples button opens the Edit Samples window where sample names types and standard concentrations can be edited see Section 7 8 4 The toggler is shown below The additional options displayed appear after clicking the right mouse button over the toggler Page Page 1 Al A2 A3 4 A5 AB A AR Edit Samples Select Non Empty Samples Toggle Sample ID Display Select Groups B5 Named On Al On All Off Edit Samples 7 4 Rotor Gene Q User Manual 11 2009 7 5 7 5 1 Rotor Gene Q User Manual Page Toggle Sample ID Display Analysis User Interface This label at the top of the toggler indicates
123. his instrument may contain infectious agents Handle such samples with the greatest of care and in accordance with the required safety regulations Always wear safety glasses 2 pairs of gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are suitably trained and not exposed to hazardous levels of infectious agents as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws DE Infekti ses Probenmaterial Proben die mit Hilfe dieses Ger tes prozessiert werden k nnen infekti se Agenzien enthalten Die Probenhand habung sollte aus diesem Grund mit gr ter Vorsicht und gem den anzuwendenden Sicherheitsbestimmungen erfolgen Es sollten immer Sicherheitsbrille zwei Paar Handschuhe und ein Laborkittel getragen werden Der Betreiber der Anlage ist f r die Gew hrleistung der Sicherheit am Arbeitsplatz verantwortlich Er hat sicherzustellen dass die Bediener des Ger tes ausreichend geschult sind und der Umgang mit infekti sen Agenzien nicht das in den Sicherheitsdatenbl ttern oder in anderen zu beachtenden Dokumenten festgelegte Ausma berschreitet Bei der Behandlung von Abluft und bei der Abfallbeseitigung sind a
124. ide to the on position A blue Standby light on the front of the Rotor Gene Q indicates that the instrument is ready for use Rotor Gene Q User Manual 11 2009 4 5 Installation Procedures 4 Double click the Rotor Gene Q Series Software desktop icon to initiate the software A Welcome window appears the first time the software is started but does not appear for subsequent software upgrades Introductory Screen and Initial Setup Welcome Welcome Before you begin the analysis software needs to know a couple of things about the type of system you are using Machine Serial Number Port COM1 v Auto Detect Run in Virtual Mode For Demonstration 1 Begin Exit Program Machine Serial Type in the serial number 6 digits which Number can be found on the back of the Rotor Gene Q Port Choose either USB or serial cable If using a serial cable select the appropriate communications port or click the Auto Detect button Run in Virtual Checking this box allows installation of the Mode for Rotor Gene Q software on a computer that demonstration is not connected to a Rotor Gene Q The software is fully functional and can simulate runs Note If this box is checked and a Rotor Gene Q is connected to the computer the following message appears before the run starts You are about to run in Virtual mode To perform a real run the setup must be changed in the Setup window see Sect
125. iers and filtering them out of subsequent HRM analysis will greatly improve the overall effectiveness of HRM analysis since analyzing poor quality PCR product will result in poor HRM results We recommend analyzing quantitative real time PCR data as follows 1 Analyze the real time data using the Quantitation option from the Analysis window If any Cy values are 30 or higher the corresponding reactions are considered to have amplified too late These samples must be analyzed with suspicion or removed from the analysis as an outlier Late amplification is usually due to too little starting template amount and or high levels of sample degradation Rotor Gene Q User Manual 11 2009 11 17 High Resolution Melt Analysis 2 Assess the end point fluorescence level If end point fluorescence in any of the amplification plots is low compared with the majority of plots in the data set omit those samples from the analysis even if their C value is less than 30 Low end point fluorescence can indicate incorrect dye amount incorrect levels of reaction components such as primers or the action of inhibitors 3 Use the Comparative Quantitation option from the Analysis window to obtain the reaction efficiency of each sample If the efficiency is not similar to other reactions in the experiment or is less than approximately 1 4 omit the reaction as an outlier Comparative Quantitation Analysis Cycling A Green Page 1 7
126. ignal and detection sensitivity A Rotor Holder that enables easy tube loading is provided with the instrument IMPORTANT To achieve maximum temperature uniformity each position in the rotor must contain a tube Filling all positions in the rotor ensures even airflow to every tube Keep a set of empty capped tubes available that can be used to fill any unused positions Insert the 72 Well Rotor Locking Ring onto the 72 Well Rotor by pushing the 3 locating pins through the outer holes of the rotor The Locking Ring ensures that caps remain on tubes during a run Rotor Gene Q User Manual 11 2009 Operating Procedures Hardware 5 Insert the assembly into the Rotor Gene Q chamber by clicking into place using the locating pin on the rotor hub To remove simply push down on the rotor hub to release and pull out 6 Close the lid and set up the run profile using the Rotor Gene Q software Rotor Gene Q User Manual 11 2009 5 7 Operating Procedures Hardware 5 3 5 8 Rotor Disc setup The Rotor Disc 72 or Rotor Disc 100 comprise 72 or 100 wells respectively in a one piece disc designed for high throughput The Rotor Disc 72 and Rotor Disc 100 do not use caps Instead Rotor Disc Heat Sealing Film is applied to the top and heat sealed using a Rotor Disc Heat Sealer The film prevents contamination by providing a strong durable and tamper proof seal Heat sealing the Rotor Disc is performed as desc
127. ilutions with 1 2 8 and 16 copies the average should be 4 copies because it is the middle of the dilution series However the arithmetic mean is 6 75 The geometric mean is 1 2 8 16 1 4 4 copies More information on geometric means can be 7 24 Rotor Gene Q User Manual 11 2009 Analysis User Interface found at http mathworld wolfram com GeometricMean html Dynamic tube normalization The Dynamic Tube option is selected by default and is used to determine the average background of each sample just before amplification commences Standard normalization simply takes the first 5 cycles and uses these as an indicator of the background level of each sample All data points for the sample are then divided by this value to normalize the data This can be inaccurate because for some samples the background level over the first 5 cycles may not be indicative of the background level just prior to amplification In contrast dynamic tube normalization uses the second derivative of each sample trace to determine a takeoff point for each sample The background level is then averaged from cycle 1 up to this takeoff cycle number for each sample This gives the most precise quantitation results Note that for some data sets background fluorescence is not consistent during the cycles before amplification commences In these cases it may be necessary to deselect dynamic tube normalization by clicking on Dynamic Tube because it c
128. ing the Loading Block 96 x 0 2 ml Tubes for PCR Tubes 0 2 ml the Loading Block 72 x 0 1 ml Tubes for Strip Tubes and Caps 0 1 ml set up with a single channel pipet the Loading Block 72 x 0 1 ml Multi channel for Strip Tubes and Caps 0 1 ml set up with a multichannel pipet the Rotor Disc 72 Loading Block for the Rotor Disc 72 or the Rotor Disc 100 Loading Block for the Rotor Disc 100 All blocks are made of aluminum and can be precooled The Loading Block 72 x 0 1 ml Tubes pictured holds 18 Strip Tubes as well as up to eight 0 5 ml tubes which can be used to prepare master mix and up to sixteen 0 2 ml tubes which can be used to set up standard curves The procedure below describes reaction setup using the 72 Well Rotor The same procedure can be used for reaction setup using the 36 Well Rotor and appropriate accessories Rotor Gene Q User Manual 11 2009 Operating Procedures Hardware 1 Place the Strip Tubes into the Loading Block and aliquot the reaction components 2 Place the Caps securely on the Strip Tubes and visually inspect to confirm a tight seal Rotor Gene Q User Manual 11 2009 5 5 Operating Procedures Hardware 5 6 Insert the Strip Tubes into the 72 Well Rotor ensuring that each tube sits correctly in place Samples will not be optimally aligned over the detection system if not placed correctly in the rotor This could result in a reduction in acquired fluorescence s
129. ings Edit Profile Channel Setup Name Source Detector Gain Create New 470nm 510nm Edit 530nm 555nm re SE 585nm 610nm Edit Gain B25nm 660nm 680nm 710hp Remove gek Reset Defaults Gain ptimisation Skip Wizard lt lt Back Next gt gt Edit Profile The Edit Profile window allows the cycling conditions and acquisition channels to be specified The initial profile shown is based on the template selected when setting up the run see page 6 1 The profile is displayed graphically The list of the segments of the profile appears below the graphical display This list can include Hold page 6 13 Cycling page 6 14 Melt page 6 17 or HRM if the instrument has a HRM channel page 6 18 Each stage of the profile can be edited by clicking on the appropriate area of the graphical display or on the name in the list and then changing the settings which appear Insert after This allows addition of a new cycle after the selected cycle Insert before This allows addition of a new cycle before the selected cycle Remove This removes the selected cycle from the profile Rotor Gene Q User Manual 11 2009 Operating Procedures Software Hold A Hold instructs the Rotor Gene Q to remain at the designated temperature for a set time To change the temperature click on the Hold Temperature button and type or use the slide bar to select the desired temperature To ch
130. ion 7 5 4 4 6 Rotor Gene Q User Manual 11 2009 Installation Procedures Begin When all the information has been entered click Begin Wait until initialization is finished which may take a few seconds If virtual mode was chosen the following message appears Rotor Gene Q Series Software x i This software will perform basic simulation of a machine For training and demonstration purposes You can disable this setting via the Setup screen accessible from the File menu If the Run in Virtual Mode box is unchecked the software initializes and opens automatically Exit Program Clicking on this button exits the program 4 6 Software version Rotor Gene Q software development is ongoing To find out your version number click on Help then About This Software Rotor Gene Q User Manual 11 2009 4 7 Installation Procedures Rotor Gene Q Pure Detection Software Version 2 0 2 Build 3 Machine Serial No 123456 Model 5 Plex Updates and additional information available at http www giagen com Warning This computer program is protected by copyright law and international treaties Unauthorized reproduction of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent possible under the law Portions of Info Zip c 1990 1999 Info ZIP Pty Ltd System Info Help This window displays
131. isc Loading Block Ensure that the Rotor Disc Loading Block is pushed in completely Rotor Gene Q User Manual 11 2009 5 9 Operating Procedures Hardware 7 To activate the sealing mechanism first press down on the blue anodized bar at the top of the Heat Sealer then push back the black catch 8 When the sealing mechanism has lowered an orange Sealing light illuminates If the Rotor Disc Loading Block is not in the correct position a warning beep sounds Rotor Gene Q User Manual 11 2009 Operating Procedures Hardware 9 When sealing is finished a beep sounds and the orange Ready light illuminates Press down on the blue anodized bar to raise and lock the sealing mechanism back in its original position Do not continue sealing for any longer than indicated by the beep or the Rotor Disc may deform 10 Slide the Rotor Disc Loading Block out of the Rotor Disc Heat Sealer Allow the film to cool for approximately 10 seconds and then gently remove the excess film 11 Remove the Rotor Disc from the Rotor Disc Loading Block Load the Rotor Disc into the rotor using the position one locator tab as a guide to the correct orientation Rotor Gene Q User Manual 11 2009 5 11 Operating Procedures Hardware This page intentionally left blank 5 12 Rotor Gene Q User Manual 11 2009 6 1 Operating Procedures Software Operating Procedures Software New runs can be set up using t
132. it as required This process may not be instantaneous as the Rotor Gene Q takes 4 seconds to acquire each point in each channel and during this time the user interface is deactivated 6 Repeat the process until the Fl is at the desired level Click on Stop If the run is still acquiring data when the Stop button is clicked the Rotor Gene Q finishes acquiring first and then stops This process can take up to 5 seconds for each acquiring channel 6 30 Rotor Gene Q User Manual 11 2009 Operating Procedures Software 6 2 5 6 2 6 New Run Wizard window 4 This window summarizes the run Check the parameters and if they are correct click Start Run You will be prompted for a file name You can also save the run settings as a template for future runs using the Save Template button New Run Wizard Summary TH NHANH Setting Green Gain 5 Rotor 36 Well Rotor Sample Layout 12 3 Reaction Volume in microliters 25 Start Run nce you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template to save settings for future runs Skip Wizard lt lt Back New Run Wizard window 5 Enter sample types and descriptions in this window while the run is in progress The functionality of this window is identical to the Edit Samples window page 7 70 Sample information may also be entered after the run has finished The Finish and Lock Samp
133. itabilities 7 77 Support 7 100 T Technical assistance 2 2 TeeChart Office 8 4 8 6 Temperature graph 7 68 Templates adding to advanced wizard 6 9 adding to quick start wizard 6 3 allelic discrimination 7 46 8 1 endpoint analysis 7 58 8 1 melt analysis 7 41 8 1 quantitation 7 30 8 1 scatter graph analysis 7 49 8 1 Thermal performance 3 1 Three step with melt 6 2 6 8 Toggler 7 3 Toolbar 7 1 Transportation A 2 Troubleshooting 12 1 HRM 12 4 Rotor Gene Q 12 3 Tube layout 7 67 Two standard curve method 7 30 Two step 6 2 6 9 U Unpacking 4 2 User assigning roles 7 86 creating account 7 82 multiple accounts 7 91 V Virtual mode 4 6 7 10 W Warnings 1 1 Waste disposal 1 7 Wrench icon 8 5 Rotor Gene Q User Manual 11 2009 Index 3 Index This page intentionally left blank Index 4 Rotor Gene Q User Manual 11 2009 www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 3865 3865 Fax 021 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Order
134. l 11 2009 High Resolution Melt Analysis ifi HRM Results HRM A HRM Page 1 Confidence AA Human SNP 1560031276 homo AA BB unknown homo AA BB unknown homo AA BR AG Human SNP 560031276 hetero AG unknown hetero AG unknown hetero AG EI GG Human SNP 1560031276 homo GG unknown homo GG unknown homo GG SNP genotyping by HRM Human SNP rs60031276 A to G substitution in the PPP1R14B gene protein phosphatase 1 regulatory inhibitor subunit 14B was analyzed on the Rotor Gene Q using 10 ng genomic DNA of different genotypes and the Type it HRM Kit available soon Homozygous wild type AA homozygous mutant GG and heterozygous AG samples are shown on H a standard normalized melt curve and B a difference plot normalized to wild type samples Genotypes for the unknown samples were assigned by the Rotor Gene Q software 11 4 Methylation analysis example In the example shown the EpiTect HRM PCR Kit was used in HRM analysis to discriminate various ratios of methylated and unmethylated DNA For technical details consult the EpiTect HRM PCR Handbook Rotor Gene Q User Manual 11 2009 11 5 High Resolution Melt Analysis 10096 methylated 0 Quantitative methylation analysis by HRM Various ratios of methylated and unmethylated DNA APC adenomatosis polyposis coli were analyzed and discriminated by HRM methylation analysis on the 11 6 Rotor Gene Q User Manual 11 2009 11 5 High Resolution Mel
135. le the sample page for the gene of interest may apply to the green channel and the sample page for the housekeeper gene may apply to the yellow channel In this example setting up sample page suitability reduces the number of analysis options available to just include those relevant for the particular assay Rotor Gene Q User Manual 11 2009 7 77 Analysis User Interface The Sample Page Suitability window is shown below Sample Page Suitability x L Sample Page Suitabilities enable you to hide sample pages when they are not relevant in the current context For example by defining a Sample Page to apply only to the Yellow channel you will not be prompted to select it during an analysis of a Green channel This feature is of particular use for users of multiplexed assavs Page Page 1 Suitabilities for Selected Page C Always display this sample page Only display this sample page when analysing idata acquired on the following channels i Green CI Yellow U Orange Red Save amp Close Help Note When setting up an assay create all of the sample pages and sample page suitabilities then save them as a template This reduces the amount of setup required for each run Groups Sample groups allow statistics to be calculated for an arbitrary collection of samples Unlike replicates which must have identical names samples can have any name can be posi
136. les button closes the screen and prevents the sample names from being modified For more information about this and other security features see Security menu Section 7 81 Rotor Gene Q User Manual 11 2009 6 31 Operating Procedures Software New Run Wizard Settings Given Conc Format D Unit Copies DI More ptions Samples Bl Edt Reset Detaut Gradient pel Er BAND Name Type Groups Given Conc Se 4 e 1 JOEE 34 Unknown Y 2 JDE E 3B Unknown Ye 3 JDE E 3C Unknown Ye 4 JDE E 3 NTC Unknown Ye 5 Unknown Ye Unknown Ye Unknown Ye Unknown Ye Unknown ad gt m Page Name Page 1 New Delete Synchronize pages Skip Wizard lt lt Back Einish Finish and Lock Samples 6 32 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 1 7 2 7 3 Analysis User Interface This chapter describes the Rotor Gene Q software user interface Workspace The workspace is the backdrop of the main window In this area raw data plots and analysis results can be opened If several windows are opened simultaneously they can be organized by clicking the Arrange button on the toolbar There are several window arrangement options available that can be selected by clicking on the down arrow next to the Arrange button I Reports Arrange Smart Tiling Tile Horizontally Tile Vertically Cascade Toolbar These buttons are sh
137. lick on Users and Passwords 7 82 Rotor Gene Q User Manual 11 2009 Analysis User Interface Users and Passwords I 21x Users Advanced Use the list below to grant or deny users access to your computer and to change passwords and other settings Users For this computer t dministrator Administrators Lj Guest STEW Guests nen NEWLAPTOP STEW Guests IWAM NEWLAPTOP STEW Guests matthew2 STEW Administrators Add Remove Properties Password For Administrator To change the password For Administrator click Set h Password Set Password Rotor Gene Q User Manual 11 2009 7 83 Analysis User Interface 3 Click the Advanced tab then click the Advanced button Users and Passwords j 2 xl Users Advanced Certificate Management Use certificates to positively identify yourself certification authorities and publishers New Certificate Certificates Advanced User Management Go Local Users and Groups can be used to perform advanced user management tasks Secure Boot Settings m It is recommended that you require users to press Ctrl Alt Delete before logging on This ensures password security and helps protect the system from harmful programs IV Require users to press Ctrl Alt Delete before logging on OK Cancel Apply 7 84 Rotor Gene Q User Manual 11 2009 4 Analysis User Interface In
138. lle gesetzlichen Regelungen zur Gesundheit und Sicherheit auf nationaler regionaler und lokaler Ebene zu ber cksichtigen Rotor Gene Q User Manual 11 2009 1 17 Safety Information WARNING AN Echantillons contenant des agents infectieux Certains chantillons utilis s avec cet instrument peuvent contenir des agents infectieux Manipuler ce type d chantillon avec le plus grand soin et en accord avec les r gles de s curit requises Toujours porter des lunettes de protection deux paires de gants et une blouse de laboratoire La personne responsable par exemple le Chef du laboratoire doit prendre les pr cautions n cessaires la s curit de l environnement du poste de travail et s assurer que les op rateurs de l instrument sont suffisamment form s et non expos s des quantit s dangereuses d agents infectieux comme d fini dans les fiches de donn es de s curit Material Safety Data Sheets MSDS ou des documents OSHA ACGIH ou COSHH L vacuation des vapeurs et d chets doit tre conforme tous r glements et dispositions l gales au plan national d partemental et local concernant la sant et la s curit Hazardous chemicals W15 Some chemicals used with this instrument may be hazardous or may become hazardous after completion of the protocol run Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary pre
139. log concentrations and C values of the standards are used to construct a model in the form y Mx B Confidence intervals for calculated concentrations We use the following confidence interval 100 1 a for an estimate of a new observation xO from the standard curve Yo Ao 5 ry 1 1 EL EE j 1 j 1 n Orr This is the confidence interval for the concentration of a single unknown Suppose now we have k further observations at x x0 and we denote their average by 0 Then 3 a CT Hrs N 90 12 rae Lo and arguments similar to above give x 2 Yo 0 D To rj Ge EN e Ei n 32 0 2 Le KN KIT Ji jk n Sex This formula determines how confidence intervals for concentrations of replicate unknowns are determined Rotor Gene Q User Manual 11 2009 B 1 Appendix B For estimation of standards a tighter confidence interval can be obtained Yo Om 5 o x 1 t 1 4 J1 L rr The implication of this formula is that adding replicates to an standard individual concentration reduces the width of the interval for all estimates as n is increased Adding a large number of replicates to an unknown reduces its uncertainty to that of a single standard The extra replicates reduce the uncertainty due to the unknown not forming part of the linear model Confidence intervals for C values We assume that error in replicate C values is linear and normally distributed
140. mples window Section 6 1 4 Normalization Normalization of EndPoint analysis data scales all signal levels to within the range of 0 10096 At least one positive and one negative control must be selected or more if analyzing multiple channels and the standards are not multiplexed More than one positive and one negative control should be run if there is a risk that a positive control may not amplify 1 For each channel all the positive controls are analyzed and the one with the highest fluorescence is set to be 100 This means that if duplicate controls are run a positive control can fail without affecting the run 2 All the negative controls are analyzed and the one with the lowest fluorescence level is set to be O96 7 54 Rotor Gene Q User Manual 11 2009 Analysis User Interface 3 The raw fluorescence values of the remaining samples are scaled relative to the highest positive control and the lowest negative control For example Sample Type Fluorescence 1 Positive control 56 3 2 Positive control 53 0 3 Negative control 4 5 4 Negative control 4 3 5 Sample 48 1 6 Sample 6 4 This run was a success as the 2 positive and 2 negative controls are close together and are outside the fluorescence values of the samples The normalized values are Sample Type Expression 1 Positive control 100 0 2 Positive control 93 3 Negative control 0 4 4 Negative control 0 0 5 Sample 84 2 6
141. n a manner not specified by the manufacturer the protection provided by the equipment may be impaired WARNING gt Risk of personal injury and material damage W7 Loose paper underneath the Rotor Gene Q interferes with instrument cooling It is recommended that the area beneath the instrument is kept free of clutter CAUTION gt Damage to the instrument C2 Always use a locking ring on the rotor This stops caps from coming off tubes during an experiment If caps come off during an experiment they may damage the chamber 1 2 If you touch the Rotor Gene Q during an experiment while you are charged with static electricity in severe cases the Rotor Gene Q may reset However the software will restart the Rotor Gene Q and continue the experiment Electrical safety Disconnect the line power cord from the power outlet before servicing Rotor Gene Q User Manual 11 2009 1 3 Safety Information WARNING Electrical hazard W8 Any interruption of the protective conductor earth ground AN lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Lethal voltages inside the instrument When the instrument is connected to line power terminals may be live and opening covers or removing parts is likely to expose live parts To ensure satisfactory and safe operation of the R
142. ncel 7 32 Rotor Gene Q User Manual 11 2009 Analysis User Interface After completing the selections the options will be checked with a check mark as shown below Relative Quantitation x Reports Export 2 Standard Curves Relative Quantitation Gene of Interest Standard Curve Normaliser Standard Curve O Calibrator Defined IV Auto shrink window 4 Click the Reports button to display the Report Browser Select the analysis with the correct name from the list Click the Show button to display the relative quantitation report The Export option exports the results to a new Excel spreadsheet If a calibrator is included the results are calculated relative to the calibrator sample which is assigned a value of 1 lol xl r Report Categories General amp Melt Curve Analysis amp Quantitation Delta Delta CT Relative Quantitation amp Comparative Quantitation CG 2 Standard Curves Relative Quantitation 2 standrard curve Relative Quant Analysis Relative Quant Standard Curve TUN 5 The concentrations as read from the standard curves for the gene of interest GOI Conc and the normalizing gene Norm Conc as well as the relative concentration Relative Conc are displayed The results can be saved as a Word file Rotor Gene Q User Manual 11 2009 7 33 Analysis User Interface 7 6 4 Preview Colou
143. ncel Help N Il 7 40 Rotor Gene Q User Manual 11 2009 Analysis User Interface This window allows genotypes to be assigned to the incidence of peaks in bins The default genotype configuration is shown in the screenshot with heterozygous samples having 2 peaks homozygous samples a peak in the first bin and wild type samples a peak in the second bin An abbreviation can be typed into the field next to the name of each genotype This is used when printing multichannel genotyping reports so that all results from multiple channels can be easily read For multiplex analysis genotypes must be set up in each channel If for example a dual channel quenched FRET analysis is run where a wild type and heterozygous genotype are expected in each channel the bin parameters must be set up for each channel The results will then be given in a multiplex report Melt analysis templates Melt analysis templates allow the user to export normalization threshold genotype and bin settings into a single met file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Settings lt none gt Import Export 7 6 6 Comparative quantitation Comparative quantitation compares the relative expression of samples to a control sample in a run when a standard curve is not available This is frequently used in microarray analysis Warton and coworkers 2004 provide an example of
144. nditions Air temperature 15 C to 30 C 59 F to 86 F in manufacturer s package Relative humidity Max 75 noncondensing Environmental 1K2 IEC 60721 3 1 class Mechanical data and hardware features Dimensions Width 37 cm 14 6 in Height 27 5 cm 10 8 in Depth without cables 42 cm 16 5 in Depth door open 56 cm 22 in Mass 12 kg 26 5 Ib standard configuration Capacity Up to 100 samples per run using a Rotor Disc 100 Software Rotor Gene Q software supplied on the installation CD provided Thermal specifications Description Specification Temperature range Ambient to 99 C Temperature accuracy 0 25 C type measured 30 seconds after clock start A 2 Rotor Gene Q User Manual 11 2009 Appendix A Temperature resolution 0 02 C smallest programmable increment Temperature uniformity 0 01 C Ramp rate gt 15 C s heating peak ramp rates air gt 20 C s cooling Optical specifications Description Specification Excitation sources High energy light emitting diodes Detector Photomultiplier Acquisition time 4s Waste Electrical and Electronic Equipment WEEE This section provides information about disposal of waste electrical and electronic equipment by users in the European Union The European Directive 2002 96 EC on WEEE requires proper disposal of electrical and electronic equipment when it reaches its end of life The crossed out wheeled bin symbol see below indi
145. ng HRM Insert before Remove Hold Temperature op deg Hold Time 2 mins D secs 6 Modify the cycling to suit the amplicon ETT HEN x A H 9 New Open Save As Help The run will take approximately 104 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Insert after HRM Insert before Remove This cycle repeats 45 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle Timed Step 60 deg 10 seconds Acquiring to Cycling 4 95 deg for 5 secs on Green I Long Range 60 deg for 10 secs MT Touchdown Rotor Gene Q User Manual 11 2009 11 13 High Resolution Melt Analysis 7 Ensure fluorescence data will be acquired Acquire data to the green channel at the end of the anneal step Acquisition Same as Previous New Acquisition Y m Acquisition Configuration Available Channels Acquiring Channels To acquire from a channel select it from the list in the left and click gt To stop acquiring from a channel select it in the right hand list and click lt To remove all acquisitions click lt lt Dye Chart gt gt Don t Acquire Help Dye Channel Selection Chart Green 470nm 510m FAM SybrGreen alexa488 JOE CalGold CalOrange TET Yakima Yellow VIC Yelow RE HEX alexa532
146. ng the following calculations Exponential amplification 10 Reaction efficiency 100 1 Optimal values for M exponential amplification and reaction efficiency are 3 322 2 and 1 respectively The reaction efficiency is displayed in the report in full and standard reports see page 7 13 and in the Standard Curve window The slope is calculated as the change in C divided by the change in log input e g copy number A 100 efficient amplification means a doubling of amplification product in each cycle resulting in an M value of 3 322 an amplification factor of 2 and a reaction efficiency of 1 Given an M value of 3 322 the calculations are as follows Exponential amplification 10 3322 2 Reaction efficiency 10173322 1 1 Rotor Gene Q User Manual 11 2009 Analysis User Interface As an alternative example an M value of 3 8 means that the reaction has an exponential amplification of approximately 1 83 and a reaction efficiency of 0 83 or 83 Offset In a formula describing the relationship between 2 variables the offset is expressed with the letter B y Mx B The offset is also sometimes referred to as the intercept B represents the C for a given concentration of 1 unit By substituting 1 into the concentration formula as shown below Cr log 1 M B The result is Cr B The intercept can change from run to run and is a less stable measurement than the gradient
147. nge Normalise to Cycling A Red Crop start cycles Crop and cycles Options This displays the drop down menu shown above which provides options for transformation of the raw data Normalise to This enables normalization of amplification data to data from a passive reference dye such as ROX acquired in another channel 7 2 Rotor Gene Q User Manual 11 2009 7 4 Analysis User Interface Crop start cycles This creates a new channel data set in which some start cycles have been removed This is useful if large jumps are observed in the initial cycles which can occur when using certain chemistries Crop end cycles This creates a new channel data set in which some end cycles have been removed Page 1 This indicates the page that is currently selected to display the raw data plots The Edit Sample window allows the creation of multiple sample definitions For example data can be viewed with varying line thickness sample definitions and other display options This is particularly useful if relative quantitation is performed in a single channel because the user can easily switch the view between the gene of interest and housekeeper samples by defining 2 sample pages Toggling samples At the right hand side of the main window is a toggler which includes a sample legend This consists of colored cells each of which corresponds to a sample in the display The toggler is used to control which samples can be seen in t
148. ngs determine how to perform the initial melt up to the denaturation temperature to determine the fluorescence level at which the reference sample has denatured Tube Position v Ramp from o0 4 to 5 ed deg c Hold for 3 mins o secs Denature Offset fo a deg c Cancel 4 Ensure that W The tube indicated in Tube Position contains a PCR product that will show a melt peak on the green channel m The final ramp temperature will not burn the sample yet will be high enough to allow it to melt W The hold time is sufficient to denature the sample W The denature offset is set appropriately The default of 0 C is appropriate for most melts Melts with very sharp transitions may require a denature offset of 0 5 C to 2 C as determined by the user to ensure that melt transition is detected You can also define a Denature step by introducing a new Hold step Click on Insert before and select New Hold at Temperature from the menu The calibration settings will appear 6 22 Rotor Gene Q User Manual 11 2009 Operating Procedures Software Hold Temperature 96 deg Hold Time 3 mins 0 secs Calibration Step M Calibration Settings Acquiring to RealTime on tube 1 Ramp from 80 to 95 and hold for 3 mins 0 secs Offset 0 deg Edit The calibration settings are synchronized with the denature settings so a change to the hold time in the Denature step will automatically update
149. nknown Ye Unknown veal gt Name Page 1 New elete Synchronize pages Skip Wizard lt lt Back Einish Finish and Lock Samples The following warning will appear Click Yes to confirm Rotor Gene Q Series Software Lock the samples Only administrators will be able to unlock them once this has been done Yes No Once the samples are locked it will not be possible to edit samples in the Edit Samples window Rotor Gene Q User Manual 11 2009 7 97 Analysis User Interface 7 9 6 Deg BER File Edit Format Security Standard Retor Style Settings Given Conc Format Unit Copies More Options Samples Bema C ID Name Type Groups Given Conc Selected 1 250bp Unknown Yes 2 250bp Unknown Yes B 3o Unknown Yes WW 4 500bp Unknown Yes 5 500bp Unknown Yes BN 65005 Unknown Yes El 7 Unknown Yes E o Unknown Yes M 3 Unknown Yes 10 Unknown Yes 11 Unknown Yes EN 12 Unknown Yes r Page Name Page 1 4 New Delete Synchronize pages Inda OK Cancel Help Samples can also be locked and unlocked in the Edit Samples window However only an administrator can unlock samples once they have been locked nat Security tor S Any unauthorized change to the file will invalidate the Run Signature Locked templates The Locked Templates folder of the Advanced wizard contains templates fo
150. nnels 7 Cycling A FAM SYER J Cyding A IOE ZT Melt A CHI 7 Melt A CH2 Fi Melt Curve Analysis Melt A CH1 Page 1 1 2 3 4 5 5 7 8 D Bark On Bank Off NamedOn AlOn All Off Edit Samples Peak Calling IV Flip sign of dF dT Twesod mes T eege L Current User CORIT198 linsonay RG Analyst SE EE Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Current User CORIT198 insongy RG Analyst Pea 78 6 Bylogging in as an administrator again RG Operator rights can be assigned to CORIT198 linsongy and the software can be launched again This time the Analysis menu and Reports button are missing and the Run menu is enabled 7 The status bar indicates that the user CORIT198 linsongy belongs to the RG Operator group 7 90 Rotor Gene Q User Manual 11 2009 Analysis User Interface E Rotor Gene Q Series Software VIRTUAL MODE melt analysis demo Fie Run Gain View Window Help AW Pll 9 vw K E 3 New Open Save Settings Progress Profile Temp F Temperature Profile Progress The run has completed Current User CORIT198 linsongy RG Operator Rotor Gene Q Series Software 4 7 Build 1 VIRTUAL MODE Curent User CORIT198 insongy RG Operator 8 If you log in as administrator and remove all groups from the user CORIT198 linsongy the following message will appear when CORIT198 linsongy opens the software Rotor Gene Q Series Software Yo
151. o the instrument C4 The Rotor Gene Q must not be used if the lid is broken or if the lid lock is damaged Make sure that the rotor and locking ring are installed correctly Only use rotors locking rings and consumables designed for use with the Rotor Gene Q Damage caused by use of other consumables will void your warranty Besch digung des Ger tes Der Rotor Gene darf nicht betrieben werden wenn die Sicherung der Haube oder die Haube selber besch digt sind Versichern Sie sich immer dass der Rotor und der Rotor Spannring korrekt eingesetzt sind Verwenden Sie nur Rotoren Rotor Spannringe und Verbrauchsmaterialien wie Probengef e mit dem Rotor Gene Q die durch den Hersteller des Ger tes zugelassen sind F r alle Sch den die aus einer Verwendung nicht zugelassener Materialien resultiert erlischt die Ger tegarantie Dommage de l appareil Le Rotor Gene Q ne doit en aucun cas tre utilis si la fermeture s curis e de l appareil ou le couvercle lui m me sont endommag s V rifier lors de chaque exp rience que le rotor et l anneau de serrage sont dans leur position correcte Utiliser exclusivement les rotors les anneaux de serrage et les tubes de r action autoris s par le constructeur de l appareil La garantie de l appareil ne couvre aucun dommage provoqu par l utilisation de mat riel autre que celui recommand par le constructeur de l appareil Rotor Gene Q User Manual 11 2009 1 25 Safety Information
152. oftware 7 80 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 9 Security menu The Rotor Gene Q software includes features that enable it to operate securely When correctly configured the Rotor Gene Q software can ensure the following Access to the Rotor Gene Q or the analysis software is restricted to user groups Modifications to run files are logged Unauthorized modifications are detected signatures Templates used to perform runs are logged Sample names are protected Integration with Windows security To provide a strong level of accountability Rotor Gene Q software does not manage security internally Accounts groups and passwords are all managed using the Windows built in security model Windows NT Security Integration allows the same password that provides access to network files and programs to control Rotor Gene Q software access leading to less administration In larger organizations for example network administrators can easily remove access to ex users due to the centralized security model For this reason setting up the Rotor Gene Q software securely primarily involves configuration of the Windows security roles according to best practices Prerequisites To use security you must be running Windows NT4 Service Pack 6 Windows 2000 or Windows XP Professional The security features cannot be used with Windows XP Home as it does not have the fine grained access model used by the softwar
153. ome the lid lock and reach inside you risk contact with parts that are hot electrically live or moving at high speed and you may injure yourself and damage the instrument Verletzungsgefahr und Besch digung des Ger tes W hrend eines Laufes oder w hrend der Rotor sich dreht darf die Haube nicht ge ffnet werden Wenn die Haube in diesen F llen gewaltsam ge ffnet wird besteht Verletzungsgefahr durch elektrische Spannungen hei e und mechanisch schnell bewegende Teile Ebenso kann das Ger t Schaden nehmen Rotor Gene Q User Manual 11 2009 1 11 Safety Information FR WARNING AN Risque de dommages corporels et mat riels L ouverture de l appareil pendant une exp rience n est pas autoris e Si l ouverture du couvercle de l appareil est forc e lors d une exp rience il y a risque de blessure par lectrocution blessure par des pi ces de l appareil chauff es tr s haute temp rature et en mouvement Lors de cette proc dure l appareil lui m me peut tre endommag Risk of personal injury and material damage W5 If you need to stop an experiment quickly turn off the power to the instrument then open the lid Let the chamber cool before reaching inside Otherwise you risk injury by touching parts that are hot Verletzungsgefahr und Besch digung des Ger tes Wenn ein Experiment pl tzlich beendet werden muss schalten Sie zuerst das Ger t am Hauptschalter aus und ffnen Sie dann die Haube La
154. ometry and this sample should be used as the standard A minimum of 3 standards with replicates should be used Importantly DNA standards used in fluorescence detection are only linear within the range of 1 100 ng ul Within this range if the concentration of DNA is halved so is the fluorescent reading The confidence intervals for any concentration outside this range are very broad due to nonlinearity of the chemistry Type of DNA measured Differences have been observed in the measurement of various forms of DNA e g genomic DNA compared to plasmid DNA Therefore only similar DNA types should be measured together and the use of plasmid DNA as a standard should be avoided when measuring genomic DNA Run setup To set up the run select Nucleic Acid Concentration Measurement from the Quick Start wizard Rotor Gene Q User Manual 11 2009 7 59 Analysis User Interface x Buick Start Advanced m A default template for s Perform Last Run measuring the ois concentration of Three Step with Melt nucleic acid using intercalating dyes ee Two Step e Quenched FRET udeic Acid Concentration Measurement v Show This Screen when Software Opens Note Ensure that a positive control such as a high concentration standard is run in tube position 1 Without a positive control the software will be unable to optimize gain settings for maximum sensitivity You will be prompted for this before ea
155. on the scatter graph Normalization A variety of options are available to options optimize the way in which the raw data plots are normalized M Dynamic Tube dynamic tube normalization M Slope Correct noise slope correction M Ignore First x cycles correction for noise in initial cycles For more details see page 7 25 Rotor Gene Q User Manual 11 2009 7 47 Analysis User Interface Genotypes This opens the Genotyping window which is used to define which genotype is detected in each channel In this window genotypes can be assigned based on the channels in which a sample reacts The channels selected will be used to label the corners of the scatter graph and will guide the user to the general area of the scatter graph in which regions should be defined Ze Genotyping x Reacting Channels Wild Type Cycling A Green Heterozygous Cycling A Green Cycling A Yellow Mutant Cycling A Yellow zi E Cancel Help Scatter Graph The scatter graph displays the relative expression of the 2 selected channels The display is normalized to account for different fold increases in each channel and log transformed to accentuate the differences in expression between samples To perform genotyping the user defines regions by clicking and dragging a selection on the graph The selection can then be labeled based on the genotypes configured in the Genotyping window 7 48 Rotor Gene Q User Manual 11 20
156. onal Functions This page intentionally left blank 8 8 Rotor Gene Q User Manual 11 2009 Maintenance Procedures 9 Maintenance Procedures Maintaining the working performance of the Rotor Gene Q is easy Optical performance is maintained by ensuring that the lenses located at both the emission and detection source are clean This is achieved by gently wiping a cotton tip applicator moistened with ethanol over the lenses Note Clean the lenses at least once a month depending on usage Wipe the rotor chamber at the same time Keep the work bench area clean and free from dust and sheets of paper The air inlet of the Rotor Gene Q is at the bottom and loose material such as paper or dust may compromise performance To avoid dust build up keep the lid of the Rotor Gene Q closed when the instrument is not in use If the rotor chamber becomes contaminated it can be cleaned by wiping the surfaces with a lint free cloth dampened but not dripping with a 0 196 v v bleach solution Wipe the chamber with a lint free cloth dampened with PCR grade water to remove traces of bleach When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Rotor Gene Q User Manual 11 2009 9 Additional Functions This page intentionally left blank 9 2 Rotor Gene Q U
157. one DE WARNING WARNUNG WARNUNG weist auf Situationen und Umst nde hin die zu einer Verletzung des Benutzers oder anderer Personen f hren k nnen N here Angaben zu der Art der Gef hrdung und der Vermeidung solcher Situationen werden in einem Textfeld wie diesem neben der Warnung gemacht Rotor Gene Q User Manual 11 2009 1 9 Safety Information WARNING AN WARNING DANGER La formule WARNING DANGER est utilis e pour avertir des situations pouvant occasionner des dommages corporels l utilisateur ou d autres personnes Les d tails sur ces circonstances sont donn es dans un encadr semblable celui ci Risk of personal injury and material damage w1 Improper use of the Rotor Gene Q may cause personal injuries or damage to the instrument The Rotor Gene Q must only be operated by qualified personnel who have been appropriately trained Servicing of the Rotor Gene Q must only be performed by QIAGEN Field Service Specialists Verletzungsgefahr und Besch digung des Ger tes Die unsachgem e Bedienung des Rotor Gene Q kann zu einer Verletzung des Benutzers oder zur Besch digung des Ger tes f hren Die Bedienung des Rotor Gene Q darf nur durch qualifiziertes Personal das entsprechend geschult wurde erfolgen Die Wartung des Rotor Gene Q darf nur durch Mitarbeiter des QIAGEN Kundendienstes durchgef hrt werden FR Risque de dommages corporels et mat riels L utilisation non con
158. ores detected 3 4 G Gain optimisation 6 11 6 24 manual 6 29 Gain settings 7 99 Genotypes allelic discrimination 7 46 endpoint analysis 7 51 7 57 melt curve analysis 7 40 scatter graph analysis 7 48 Groups 7 78 H Hold 6 13 HRM advanced wizard 6 9 analysis 7 61 11 1 11 19 cycle 6 18 guidelines 11 7 kits 11 3 methylation analysis 11 5 quick start wizard 6 3 real time PCR 11 17 sample preparation 11 9 SNP genotyping 11 3 software 11 9 troubleshooting 12 4 Hybridisation 6 17 Ignore first 7 26 7 47 Installation 4 1 grounding requirements 4 1 hardware 4 3 PC requirements 4 1 power requirements 4 1 Rotor Gene Q User Manual 11 2009 Index 1 Index software 4 4 Intended use 2 3 L LinReg exporting to 7 8 Loading Block 5 4 Lock samples 7 96 templates 6 9 7 98 Locking Ring 36 Well Rotor 5 1 72 Well Rotor 5 2 Rotor Disc 100 5 2 Rotor Disc 72 5 2 M Machine options 7 64 Maintenance 9 1 advanced wizard 6 9 Melt 6 17 Melt curve analysis 7 37 bins 7 39 peaks 7 39 Melt curve results window 7 40 Menu analysis 7 11 display options 7 80 file 7 5 gain 7 99 help 7 100 run 7 62 security 7 81 view 7 63 window 7 100 Messages 7 65 N Noise slope correction 7 25 7 47 Normalization 7 2 dynamic tube 7 25 7 47 endpoint analysis 7 54 Nucleic acid concentration measurement 6 3 Nucleic Acid Concentration Measurem
159. ortcuts to frequently used operations These operations can also be accessed from the drop down menus ECH Mew Open Save AE E Start Pause Stop View d d a ul Ki Ey A e Settings Progress Profile Temp Samples Analysis Reports Arrange 9 Help Channels 2 Cycling A Green Cycling A velo 27 Cycling A Orange 27 Cycling A Red View raw channels Click on these buttons to view the raw unanalyzed data from particular channels in the run Channels Cycling 4 Green Cycling A Yellow Cycling A Orange Cycling A Red Rotor Gene Q User Manual 11 2009 7 1 Analysis User Interface When viewing this data a number of options are available to change the data presentation The raw data may also be transformed to facilitate different types of analysis Adjust Scale To select Adjust Scale click the right mouse button over the appropriate window Adjust Scale brings up a window in which a scale can be specified Adjust Scale X Minimum o al x Autoscale Autoscale attempts to fit the scale to the maximum and minimum readings of the data Default Scale Default Scale resets the scale to display from O to 100 fluorescence units Spanner wrench See Section 8 5 for more information Icon EI 5 10 15 gt 3 30 35 40 45 Adjust Scale Aulo Scale Defaut Scale Options Page 1 H Normalise to Cycling A Yellow Normalise to Cycling A Ora
160. otor Gene Q follow the advice below M The line power cord must be connected to a line power outlet that has a protective conductor earth ground Do not adjust or replace internal parts of the instrument Do not operate the instrument with any covers or parts removed If liquid has spilled inside the instrument switch off the instrument disconnect it from the power outlet and contact QIAGEN Technical Services If the instrument becomes electrically unsafe prevent other personnel from operating it and contact QIAGEN Technical Services the instrument may be electrically unsafe when m Itor the line power cord appears to be damaged m It has been stored under unfavorable conditions for a prolonged period m It has been subjected to severe transport stresses WARNING Electrical hazard W9 The instrument has an electrical compliance label which AN indicates the voltage and frequency of the power supply as well as fuse ratings The equipment should only be operated under these conditions 1 4 Rotor Gene Q User Manual 11 2009 WARNING A Safety Information Electrical hazard w10 Voltage selection may only be changed by your sales distributor or authorized personnel Users should not attempt to change voltage selection Doing so may void the warranty WARNING A Electrical hazard w11 Do not change the voltage selector switch setting while the instrument is connected to the mains
161. ould result in less precise quantitation Noise slope correction The background fluorescence Fl of a sample should ideally remain constant before amplification However sometimes the Fl shows a gradual increase or decrease over several cycles due to the chemistry used This produces a skewed noise level Noise slope correction uses a line of best fit to determine the noise level instead of an average and normalizes to that line Selecting this option by clicking the Slope Correct button can improve data from replicates if sample baselines are noticeably sloped Noise slope correction improves the data when raw data backgrounds are observed to slope upward or downward before the takeoff point Cr Rotor Gene Q User Manual 11 2009 7 25 Analysis User Interface Ignore First The fluorescence signal from the first few cycles in a run may not be representative of the rest of the run For this reason better results may be achieved if the first few cycles are ignored Up to 10 cycles can be ignored However if the first cycles look similar to subsequent cycles better results will be achieved by deselecting Ignore First because the normalization algorithm will have more data to work with Outlier Removal To distinguish between minor changes in fluorescence and genuine reactions in no template controls NTCs 2 measures are provided NTC Threshold and Reaction Efficiency Threshold The NTC Threshold is recommended for mos
162. owing message appears when the file is reopened Bad Run File Signature The loaded run file contains a signature which does not match the file contents This means the file has either been corrupted or tampered with since it was written by the Rotor Gene O Series Software Run file signing ensures the integrity of your run results Information about a run s signature can be found in the Run Info window Rotor Gene Q User Manual 11 2009 7 95 Analysis User Interface E Rotor Gene Q Series Software VIRTUAL MODE no signature hrm demo analysis Fie Analysis Run Gain View Security Window Help FA A gt X3 3 A a Z led HO mn B WD GB New Open Save 5 Help Settings Progress Profile Temp Samples Analysis Reports Arrange Channels 7 Cyding A Green HRM A HRM Page Page 1 General Machine Options Messages Channels T General Information Run Signatures are stored within all newly saved runs These signatures like a wax E seal on a document guarantee that no changes have been made outside the software If a file is tampered with the signature becomes invalid Run Signature There is no signature There is no signature available for this run Runs saved in previous versions may not be signed This means the file contents can not be guaranteed as free from unauthorised modification Bank On Bank Off NamedOn AllOn All Off Edit Samples Rotor Gene Series Software 4 7 Build 1 VIR
163. ple several users are using the same instrument Rotor Gene Q User Manual 11 2009 6 3 Operating Procedures Software 6 1 1 Rotor selection In the next window select the rotor type from the list Check the Locking Ring Attached checkbox and then click Next br Quick Start 1 Rotor Selection 2 Confirm Profile m Rotor Type 36 Well Rotor 72 Well Rotor M Locking Ring Attached Cancel 6 4 Rotor Gene Q User Manual 11 2009 Operating Procedures Software 6 1 2 Confirm profile The cycling conditions and acquisition channels of the template chosen are imported These can be altered using the Edit Profile window see Section 6 2 4 To initiate a run click the Start Run button It is also possible to save the template before starting the run by clicking on Save Template Leem 1 Rotor Selection 2 Confirm Profile A H 9 New Open SaveAs Help The run will take approximately 77 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Insert after Melt Insert before Remove Hold Temperature ce deg Hold Time ES eee Eal secs lt Back Save Template Start Hun Rotor Gene Q User Manual 11 2009 6 5 Operating Procedures Software 6 1 3 Save run After clicking the Start Run button the Save As window appears The run can be saved in the user s desired lo
164. preamplified PCR product to place in position 1 of the rotor This sample should contain the same PCR product as the samples of interest and a detection chemistry for monitoring PCR product dissociation M An optical denature profile A new profile can be created or an existing profile can be edited see details below An Optical Denature Cycle appears almost identical to other cycles The principal differences are the melt step automatically inserted at the beginning of the profile and the sharp profile of the denature step during cycling The Optical Denature Cycle does not require defined hold times as the dissociation of the product is monitored at each cycle To perform this technique the following information about the run is required M The initial denaturation temperature This is the same temperature as the Denature step in a standard cycling profile M The tube position of the PCR sample that will produce a melt curve on the green channel M An Optical Denature Cycling profile must be defined Create a new Optical Denature Cycle as follows 1 Open the Edit Profile window Then click on New In the window that appears click the Insert after button Rotor Gene Q User Manual 11 2009 Operating Procedures Software and select New Cycling from the menu Select one of the temperature steps by clicking on the graph In the drop down menu change from Timed Step to Optical Denature A default profil
165. quire a license Hi Res Melting Mark under license from Idaho Technology Inc for applicable countries 2009 QIAGEN all rights reserved Contents Contents 1 Safety Information 1 1 1 1 Proper use 1 2 1 2 Electrical safety 1 3 1 3 Environment 1 5 1 4 Biological safety 1 5 1 5 Chemicals 1 7 1 6 Waste disposal 1 7 1 7 Mechanical hazards 1 8 1 8 Heat hazard 1 9 1 9 Translations of warnings and cautions 1 9 1 10 Symbols on the Rotor Gene Q 1 26 2 Introduction 2 1 2 1 About this user manual 2 1 2 2 General Information 2 2 2 2 1 Technical assistance 2 2 2 2 2 Policy statement 2 2 2 2 3 Version management 2 2 2 3 Intended use of the Rotor Gene Q 2 3 3 General Description 3 1 3 1 Thermal performance 3 1 3 2 Optical system 3 3 4 Installation Procedures 4 1 4 1 Requirements 4 4 2 Unpacking the Rotor Gene Q 4 2 Rotor Gene Q User Manual 11 2009 Contents 1 Contents 4 3 4 4 4 5 4 6 4 7 5 1 5 2 5 3 6 6 1 6 1 1 6 1 2 6 1 3 6 1 4 6 2 6 2 1 6 2 2 6 2 3 6 2 4 6 2 5 6 2 6 7 7 1 7 2 7 3 7 4 7 5 Contents 2 Accessories Hardware installation Software installation Software version Updating software Operating Procedures Hardware Rotor types Reaction setup Rotor Disc setup Operating Procedures Software Quick Start wizard Rotor selection Confirm profile Save run Sample setup Advanced wizard New Run Wizard window 1 New Run Wizard window 2 New Run Wizard window
166. quisition and sample definitions from the last run open in the software Perform Last Run L 1 Three Step with Melt ee 2 Two Step ee Quenched FRET ae Nucleic Acid Concentration Measurement d HRM d Other Runs e J Open A Template In Another Folder M Show This Screen When Software Opens For information about the templates provided see Section 6 1 and Section 6 2 New Run New This initiates the run setup using the selected template Cancel This closes this window Help This opens online help Show This Screen If this box is checked the New Run When Software window is displayed when the software is Opens started 7 6 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 5 2 Open and Save Open This opens a previously saved Rotor Gene Q run file rex or Rotor Gene Q run archive rea file Open Recent This displays the last 4 files that have been opened or saved Save This saves any changes that have been made to a run file New Open Open Recent VW Save Import Date From Previous Run Template Reports Run Archive LIMS Export Ap Excel Data Sheet Exit Excel Analysis Sheet LinReg Export Format Matlab Export Save As Use this function to save the run file or data in various formats The options are listed below Run File This saves a copy of the file The user can chang
167. r Gene Q User Manual 11 2009 Analysis User Interface ERotor Gene Q Series Software VIRTUAL MODE Three Step with Melt 2008 11 26 1 G IE File Run Gain View Window Help BOW PUB o 2 9 S Ww C Wew Open Save Start Pause Stop Help Settings Progress Profile Temp Samples Arrange ucc A 9 bo om Run Settings lol xi General Machine Options Messages Channels Tube Layout Security Run Wizard Advanced amp Security Audit Trail Summary Saved At 26 11 2008 2 47 45 PM By CORITOQ1 RGQ User 2 Experiment Started At 26 11 2008 2 47 46 PM By CORITOOT RGQ User 2 Saved At 26 11 2008 3 36 00 PM By CORIT001 RGQ User 2 Saved At 26 11 2008 3 36 12 PM By CORIT001 RGQ User 2 E Security Audi Trail Detail Saved At 26 11 2008 2 47 45 PM By CORIT001 RGQ User 2 S 1 5 21 1177238915 19 0048 0049 Experiment Started At 26 11 2008 2 47 46 PM By CORIT001 RGQ User 2 5 1 5 21 117 e Saved At 26 11 2008 3 36 00 PM By CORIT001 RGQ User 2 S 1 5 21 1177238915 19 Saved At 26 11 2008 3 36 12 PM By CORITOO1 RGQ User 2 S 1 5 21 1177238915 195 Close Er Warnings NE 15 x Operator skipped end of cycle for Cycling Repeat 25 Premelt Conditioning Rotor Gene D Series Software 1 7 Build 94 VIRTUAL MODE Current User CORITOO1 RGQ User 2 AG Operator This can be used to monitor who has modified the contents of a file The Security Audit Trail Detail contains more detail s
168. r QIAGEN kits and assays which cannot be modified by the user To use these templates the Rotor Gene Software must be installed with the Force Authentication through Windows NT logon option For a detailed description of the functionalities of these templates and the requirements of the different assays please refer to the corresponding kit handbooks 7 98 Rotor Gene Q User Manual 11 2009 Analysis User Interface 7 10 It is not currently possible for the user to create read only template files using the Rotor Gene Q software However if desired it can be specified as a requirement that all runs are performed using a specific template file In order to ensure read only access to this template it should be stored on a network drive where users cannot modify data Users can still run and modify their own profiles while the template on a network drive such as this is protected In order to track which template has been used the Rotor Gene Q software stores the name of the template file that was run This information can be accessed by clicking the Settings button which then enables the Run Settings window to be displayed The template information is stored in Other Run Information Other Run Run file has a valid signature Information Template was signed Using 72 well carousel Channels saved for this run Created with template CADual Labeled Probes ret Run is currently m progress Started at 16 02 2
169. r Replicate Name GOI Conc GOI Count Norm Conc Norm Count Relative Conc Calibrat 000086 1 288 3 1 311 3 1 00 Yes ES 1976356 637 3 1 01 311 3 634 3 321 3 82 3 40 3 Delta delta C relative quantitation The delta delta C method enables relative gene expression analysis It is described by Livak and Schmittgen 2001 This method does not require standard curves to be included in each run Each sample is first normalized for the amount of template added by comparison with the normalizing gene These normalized values are further normalized relative to a calibrator treatment The calibrator could be for example wild type untreated control or time zero samples It is essential that the amplification efficiencies of the gene of interest and the normalizing gene are identical and that this is validated according to the guidelines of Livak and Schmittgen It is essential that the sample names are defined correctly in the Edit Samples window with the same samples labeled identically in each composite quantitation analysis 1 Analyze the data using Quantitation It is not necessary to run a standard curve once validation has been performed 2 From the Other tab in the Analysis window select Delta Delta C Relative Quantitation Select New Analysis Livak K J and Schmittgen T D 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2 delta delta C
170. rd 20 92 300000 298898 0 4 9 10S Standard 21 04 300000 275802 81 10 3105 Standard 24 20 30000 30286 10 2412 0 07 23 94 24 29 32005 25827 39661 1 10S Standard 24 06 30000 33076 109 12 305 Standard 24 09 30000 32530 8 4 13 3004 Standard 2751 3000 2987 DS 27 42 0 09 27 20 27 63 3188 2427 4190 14 3104 Standard 2740 3000 3223 7 4 15 3104 Standard 2734 3000 3367 122 16 3403 Standard 30 76 300 308 25 30892 0 16 30 52 31 32 274 189 397 1 3003 Standard 31 08 300 246 181 2103 Standard 3053 300 273 9 0 In the Quantitation Results window the results from the run are summarized in a table Clicking the right mouse button and selecting Export to Excel exports the table to Excel Excel opens automatically To copy the data into an existing spreadsheet choose the Copy option instead open the spreadsheet then select Paste 7 20 Rotor Gene Q User Manual 11 2009 Analysis User Interface The Quantitation Results window includes the following columns Var Rep Cr Rep C Std Dev Rep Cr 95 Cals Rep Calc Conc The percentage variation between the calculated and the known concentration Var Abs Calculated Given 1 The average Cr of all samples with the same name as this sample The standard deviation of the C value of all samples with the same name as this sample A Cr range that statistically accounts for 95 of the variation in the Cy value
171. rers may autofluoresce which could affect the reliability of results In addition tubes from alternative manufacturers can vary in length and thickness resulting in misalignment of the optical path of the Rotor Gene Q and the reaction in the tube 7 EEE Rotor Gene Q User Manual 11 2009 5 Operating Procedures Hardware 72 Well Rotor The 72 Well Rotor is blue in color The 72 Well Rotor and 72 Well Rotor Locking Ring are used with Strip Tubes and Caps 0 1 ml which can be used for volumes as low as 10 ul The caps provide a safe and reliable seal Rotor Disc 72 Rotor The Rotor Disc 72 Rotor is dark gray in color The Rotor Disc 72 Rotor and Rotor Disc 72 Locking Ring enable use of the Rotor Disc 72 The Rotor Disc 72 is a disc with 72 wells for high throughput use To seal the Rotor Disc 72 a clear polymer film is applied to the top and heat sealed The film is quick to apply and prevents contamination by providing a strong durable and tamper proof seal For more information on the Rotor Disc 72 see Section 5 3 Rotor Disc 100 Rotor The Rotor Disc 100 Rotor is gold in color The Rotor Disc 100 Rotor and Rotor Disc 100 Locking Ring enable use of 5 2 Rotor Gene Q User Manual 11 2009 Operating Procedures Hardware the Rotor Disc 100 The Rotor Disc 100 is a disc with 100 wells for high throughput use The Rotor Disc 100 is the rotary equivalent of a 96 well plate but with an addi
172. review window After the report has been generated the buttons on the top of the Preview window can be used to print save or e mail the report or export it to Word Print Save bs Email To Word Standard Curve Std Curve This button opens the Standard Curve window By default this window is opened when an analysis is opened If you close the window it can be reopened using this command Rotor Gene Q User Manual 11 2009 7 13 Analysis User Interface ERI Standard Curve Cycling A Gre 10 xl Cycling amp Green Page 1 R 0 99969 R 2 0 99939 M 3 292 B 38 949 Efficiency 1 DI Export Graph Standard Error Mean Overlay v Show Stats zoom Qut 1c Return to Default Position 004 10 95 10 06 10 07 oncentration The values on the standard curve are recalculated dynamically as the threshold level is varied by clicking and dragging the threshold line in the main window Blue dots on the curve represent the samples that have been defined as standards and red dots represent the unknown sample data points Note If redefining standards to recalculate the standard curve toggling the standard sample visibility to off using the toggler on the right of the screen will remove it from the standard curve calculation Removing standards from the graph to increase the R 2 value is not scientifically valid A failed standard is an indication that the samples may also have failed and
173. ribed below IMPORTANT Please read the Product Sheet supplied with the Rotor Disc Heat Sealer before beginning this procedure 1 Switch on the Rotor Disc Heat Sealer using the switch located on the back at the left hand side A red Power light illuminates The Rotor Disc Heat Sealer takes approximately 10 minutes to reach operating temperature when a green Ready light illuminates Note Once the Rotor Disc Heat Sealer is ready it is safe to leave it running constantly 2 Insert the Rotor Disc into the Rotor Disc Loading Block using the position one tab on the Rotor Disc and the tube guide holes on the Rotor Disc Loading Block 3 Setup reactions in the Rotor Disc by manual pipetting or using the QIAgility automated liquid handling system Rotor Gene Q User Manual 11 2009 Operating Procedures Hardware 4 Remove the central portion from one sheet of Rotor Disc Heat Sealing Film by slightly folding the film in half pinching the center piece and carefully tearing it out 5 Place the film over the Rotor Disc in the correct orientation as shown by the SIDE UP label Ensure that the SIDE UP label is positioned at the bottom of the Rotor Disc Loading Block The central hole in the film should slide easily over the cylinder of the Rotor Disc Loading Block and onto the top of the Rotor Disc 6 Slide the assembly into the Rotor Disc Heat Sealer using the guide rails on the side of the Rotor D
174. rmalsed Graph Difference Graph Normalised minus mutation 4 Bank On Bank Off NamedOn AllOn All Off Edit Samples r Normalisation Regions Leading Range Stat 24 End 828 2 Trailing Range Stat e93 End ar H r Confidence Percentage Threshold 90 4 Rotor Gene Q Series Software 1 7 Build 94 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis 6 Genotypes will be called automatically by the software in the Results window A confidence value is provided as an integrity check of autocalled results The threshold value above which autocalls are made can be edited Samples that fall below the set threshold will be marked as a variation for closer investigation or retesting ini x Nermaisation Regions Leading Range Confidence i d mutation 100 00 Start 824 heterozygote 100 00 End 82 8 mutation 95 72 heterozygote 97 58 Trailing Range heterozygote 37 80 Start 189 3 wild type 100 00 Wess FE wild type 33 85 End 83 heterozygote 98 91 heterozygote 96 23 r Confidence Percentage gt wild type 9323 Threshold 50 4 wild type 97 59 Rotor Gene Q User Manual 11 2009 11 23
175. rument is kept free of clutter Verletzungsgefahr und Besch digung des Ger tes Lose Papierseiten oder andere d nnen Materialien unterhalb des Rotor Gene Q k nnen die Instrumentenk hlung st ren Es wird empfohlen solche Materialien aus dem Bereich rund um den Rotor Gene zu entfernen FR WARNING A Risque de dommages corporels et mat riels Des feuilles de papier volant ou quivalent se trouvant sous le Rotor Gene Q peuvent perturber le m canisme de refroidissement de l appareil Il est donc conseill de retirer tout papier pouvant se trouver proximit de l appareil Electrical hazard W8 Any interruption of the protective conductor earth ground lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Lethal voltages inside the instrument When the instrument is connected to line power terminals may be live and opening covers or removing parts is likely to expose live parts Rotor Gene Q User Manual 11 2009 1 13 Safety Information DE Gef hrdung durch Elektrizit t Das Ger t muss zum Betrieb immer geerdet sein Es ist verboten die Schutzleiter im Ger t oder in der Netzzuleitung zu trennen oder zu entfernen Gef hrliche Spannung im Ger t Auch in ausgeschaltetem Zustand kann an einigen Stellen im Ger t Netzspannung anliegen wenn das Ger t am Stromnetz
176. s Rel Other Quantitation Melt E Cycling A FAM mA RT Eeer Show Hide Auto shrink window Given Conc This shows the concentration for each of the standards The units can be defined as a decimal or log number If the standards are a dilution series it is only necessary to type the first 2 standards By pressing ENTER the program automatically adds the next logical dilution in the series Line style The style of the line can be modified to improve readability of graphs on black and white printers Certain lines can be emphasized by modifying their style To access this feature click on the right arrow button next to the Edit button D d Edit Reset Defau Gradient Rotor Gene Q User Manual 11 2009 7 75 Analysis User Interface Multiple row entry Sample type hotkey Save it reuse it Rotor Style The toolbar will show the default style Solid This can be changed to Dashed Dotted Hairline Thin or Thick When finished click the left arrow button to return to the Edit Reset Default and Gradient view Hc If the same information needs to be entered in several rows at once select all the rows then begin to type The information will be entered into each row This also works for selecting sample types choosing colors or entering concentrations To quickly select a sample type enter the first letter of its name For example to set 5 samples to b
177. s 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 m Sample amp Assay Technologies
178. s authorized in writing by an officer of the Company Read out devices interfacing devices and associated software will be warranted only for the period offered by the original manufacturer of these products Representations and warranties made by any person including representatives of QIAGEN which are inconsistent or in conflict with the conditions in this warranty shall not be binding upon the Company unless produced in writing and approved by an officer of QIAGEN Rotor Gene Q User Manual 11 2009 D 1 Appendix D This page intentionally left blank D 2 Rotor Gene Q User Manual 11 2009 Index Index A Acquisition 6 15 Adjust scale 7 2 Advanced wizard 6 7 Allelic discrimination 7 44 Audit trails 7 92 Auto find threshold 7 20 Autoscale 7 2 AutoStat 7 23 C Calibrator replicate 7 43 Cautions 1 1 Channels 3 4 7 65 Comparative quantitation 7 41 Concentration analysis 7 58 Correlation coefficient 7 15 Crop cycles 7 3 C calculation 7 18 Cycling 6 14 D Default scale 7 2 Delta delta C relative quantitation 7 34 Detection parameters 3 4 Dynamic tube normalization 7 25 7 47 Edit profile window 6 5 6 12 Edit samples window 6 7 6 31 7 70 rotor style 7 76 Efficiency 7 14 7 28 Empty run 6 8 EndPoint analysis 7 49 controls 7 53 Excitation parameters 3 4 Exponential amplification 7 28 Export data 8 5 graphs 8 2 native format 8 4 to LinReg 7 8 F Fluoroph
179. s have been performed in the same run it is possible to overlay the standard curves in the same window This is useful for graphically viewing the difference between different thresholds This feature is shown in the screenshot below Rotor Gene Q User Manual 11 2009 7 15 Analysis User Interface IS standard Curve Cycling A Green Page 1 zigi xi Cycling amp Green Page 1 R 0 99969 R 2 0 99939 M 3 292 B 33 949 Efficiency 1 01 Export Graph Standard Error Mean Overlay lv Cycling 4 Yellow Page 2 v Show Stats Cycling A Orange Page 3 Zoom Out Cycling 4 Red Page 4 Return to Default Position FE TT P e DEES 10 00 10 01 10 02 10 03 10 04 10 05 10 06 10 07 Concentration Standard curve calculation conc Cr and C4 are 2 versions of the equation used to relate C values and concentrations In publications the C formula is most commonly used The standard curve can be either Floating or Fixed If Floating an optimal equation for the standard curve is calculated each time the threshold is moved in the main window If Fixed the equation does not change because it has been imported from another run Standard Curve conc 10 0 304 CT 11 832 CT 3 292 loglconc 38 343 Type Floating Import Curve Reset Import Curve Importing a standard curve allows estimation of concentrations when a standard curve is not available in a par
180. s select this option 1A 1B 1C should be selected when samples were loaded in adjacent O 1 ml Strip Tubes using a multichannel pipet with 8 channels The A1 A2 A3 layout may be selected if appropriate 6 10 Rotor Gene Q User Manual 11 2009 Operating Procedures Software New Run Wizard This box displays 1 This screen displays miscellaneous options for the run Complete the fields help on elements in clicking Next when you are ready to move to the next page perator your mouse over the item for help Y ou Notes can also click on a combo box to display help about its available settings Reaction Volume pL Sample Layout 11 2 3 Skip Wizard lt lt Back Next gt gt 6 2 3 New Run Wizard window 3 In this window the Temperature Profile and Channel Setup can be modified If the Edit Profile button is clicked the Edit Profile window appears enabling alteration of cycling conditions and selection of acquisition channels Section 6 2 4 After setting up the profile click the Gain Optimisation button to bring up the Gain Optimisation window see page 6 24 Rotor Gene Q User Manual 11 2009 6 11 Operating Procedures Software 6 2 4 New Run Wizard Temperature Profile the wizard For help on an item hover your mouse over the item for help Y ou can also click on a combo box to display help about its available sett
181. s the run will take to finish amp Profile Progress Skip Skip allows any steps of the profile to be skipped Rotor Gene Q User Manual 11 2009 7 69 Analysis User Interface Add 5 Cycles Add 5 Cycles adds 5 repeats to the current cycling step 7 8 4 Edit Samples icix File Edit Format Security Standard Rotor Style Settings Given Conc Format 122457 Unit lege Y More Options Samples gl gt Edit Reset Default Gradient HS zs ID ame T ype Groups Given Conc Selected R 1000 Standard 1 000 Yes 5 R 1000 Standard 1 000 Yes 83 A100 Standard 100 Yes A4 R100 Standard 100 Yes A5 R10 Standard 10 Yes 7 AB R10 Standard 10 Yes A R1 Standard 1 Yes Wan n Standard 1 Yes I RNTC NTC Yes RNTC NTC Yes Eu B3 11000 Standard 1 000 Yes Bb 1000 Standard 1 000 Yes Page Name Pe gt New GES Synchronize pages Undo DK Cancel Help Click on the Samples button to bring up the Edit Samples window The Edit Samples window can also be accessed by right clicking over the sample list on the right of the screen This window has identical functionality to the Edit Samples window in the wizards except that the toolbar functions are also available in the File and Edit menus Four menus appear at the top of the window File Edit Format and Security The File menu is used to create a new blank Edit Samples window
182. ser Manual 11 2009 10 10 1 10 2 Optical Temperature Verification Optical Temperature Verification Optical Temperature Verification OTV is a method that verifies the in tube temperature in a Rotor Gene Q Validation of in tube temperature can be an important procedure in certified laboratories OTV is performed using a Rotor Disc OTV Kit see Appendix C OTV principle OTV uses the optical properties of 3 thermochromatic liquid crystals TLC as absolute temperature references When heated TLCs change from opaque to transparent at very precise temperatures 50 C 75 C and 90 C TLCs do not themselves fluoresce Therefore it is necessary to cover the excitation source with a fluorescent insert so that the TLC transition points can be detected by the Rotor Gene Q optical system TLCs that are below their transition temperature are opaque and reflect light Some of the reflected light scatters towards the detector increasing fluorescence When the in tube temperature reaches the TLC transition point the TLC becomes transparent and light passes through the sample rather than being reflected toward the detector resulting in a decrease in fluorescence The change in fluorescence is used to determine the precise transition temperature of each TLC The transition temperature is compared with the temperature reported by the factory calibration file for the OTV Rotor Disc to verify whether the Rotor Gene Q is within temperature
183. so should be included in the results Efficiency This is the reaction efficiency of the run This value is discussed in more detail on page 7 28 7 14 Rotor Gene Q User Manual 11 2009 Analysis User Interface R 2 value The R 2 value or R value is the correlation percentage of the data that is consistent coefficient with the hypothesis that the standards form a standard curve If the R value is low then the standards do not easily fit onto a line of best fit This means that the results i e the calculated concentrations may not be reliable A good R value is approximately 0 999 Note It is possible to achieve a high R 2 value with a poor standard curve if an insufficient number of standards have been run The R 2 value improves as the number of standards decreases For a more accurate indication of the reliability of the results use the confidence intervals on the calculated concentrations as a guide R value square The R value is the square root of the R 2 root of value In general the R 2 value is more correlation useful for determining correlation coefficient M and B The slope M and the intercept B of the standard curve are automatically calculated using the formula y Mx B and shown in the Standard Curve window Export Graph Clicking the right mouse button over the standard curve displays the option to export the graph see Section 8 4 Overlay When multiple quantitation run
184. sque de feu Lors du nettoyage du Rotor Gene Q avec un d sinfectant base d alcool laisser le couvercle du Rotor Gene Q ouvert pour permettre aux vapeurs inflammables de s vaporer Nettoyer le Rotor Gene Q uniquement quand les composants de la table de travail ont refroidi WARNING AN Moving parts w17 To avoid contact with moving parts during operation of the Rotor Gene Q the instrument must be operated with the lid closed Bewegliche Ger teteile W hrend des Ger telaufs ist jeglicher Kontakt mit den beweglichen Ger teteilen zu vermeiden das Ger t darf ausschlie lich bei geschlossener T r in Betrieb genommen werden FR El ments mobiles Lorsque le Rotor Gene Q est en marche toujours fermer le couvercle de l instrument afin d viter tout contact avec les l ments mobiles de l appareil 1 20 Rotor Gene Q User Manual 11 2009 Safety Information WARNING Risk of personal injury and material damage W18 Open and close the lid of the Rotor Gene Q carefully to A avoid trapping fingers or clothing DE Verletzungsgefahr und Besch digung des Ger tes Offnen und schlie en Sie die Haube des Rotor Gene Q vorsichtig so dass weder Finger noch Kleidungsst cke eingeklemmt werden FR Risque de dommages corporels et mat riels WARNING AN Ouvrir et fermer le couvercle du Rotor Gene Q avec pr caution afin d viter de se pincer les doigts ou de coincer des v tement entre l
185. ssen Sie die Reaktionskammer abk hlen bevor sie hineinfassen da sonst Verletzungsgefahr durch hei e Oberfl chen und Teile besteht Risque de dommages corporels et mat riels Si une exp rience doit tre interrompue de fa on subite veuillez d abord teindre l appareil en utilisant l interrupteur principal d alimentation Lorsque l appareil est teint le couvercle de l appareil peut alors tre ouvert Laissez refroidir la chambre int rieure de l appareil pour viter toute br lure au contact de surfaces ou de pi ces tr s chaudes WARNING AN Risk of personal injury and material damage W If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Rotor Gene Q User Manual 11 2009 Safety Information DE Verletzungsgefahr und Besch digung des Ger tes Wenn das Ger t in einer von den Herstellerangaben abweichenden Art und Weise verwendet wird k nnen Schutzvorkehrungen am Ger t in ihrer Wirksamkeit eingeschr nkt sein FR Risque de dommages corporels et mat riels Si l appareil n est pas utilis selon les recommandations du constructeur les mesures de protection fournies par l appareil peuvent perdre leur efficacit WARNING AN DE Risk of personal injury and material damage W7 Loose paper underneath the Rotor Gene Q interferes with instrument cooling It is recommended that the area beneath the inst
186. standard melt the temperature is increased by increments of 1 C waiting for 5 seconds before each acquisition The Rotor Gene Q can be configured to perform melts in 0 02 C increments The minimum hold time between temperature steps varies depending on the number of degrees between each step High Resolution Melt High resolution melt HRM analysis characterizes double stranded DNA samples based on their dissociation melting behavior It is similar to classical melting curve analysis but provides far more information for a wider range of applications Samples can be discriminated according to sequence length GC content or strand complementarity down to single base pair changes HRM analysis can only be performed on instruments that have HRM hardware and software installed Data is acquired using specialized HRM sources and detectors HRM analysis also includes the option to perform Gain Optimisation just before the Melt begins After performing HRM the data can be analyzed with HRM analysis software Section 11 Optical Denature Cycling Optical Denature Cycling is an exciting technique available on the Rotor Gene Q which performs real time melt analysis to determine the melt peak of a reference sample This indicates PCR product denaturation with higher precision than setting a particular denature temperature for a hold time To perform this technique simply place a reference tube of PCR product in tube position 1 of the ro
187. sult is displayed in scientific notation in the Rep Conc column of the Comparative Quantitation Results window Allelic discrimination Allelic discrimination uses real time kinetic data from 2 or more channels to genotype samples To perform this analysis select Other and then Allelic Discrimination in the Analysis window When performing allelic discrimination it is not sufficient to double click on one channel to analyze because this analysis is performed using multiple channels simultaneously To perform this analysis either hold down CTRL and click to highlight each channel you wish to analyze or drag the mouse pointer over these channels Once the desired channels have been highlighted click Show The list will update to show all the channels on one line with a checkmark beside them This indicates that they will all be used in one analysis To remove one or more of these channels right click on the analysis and select 7 44 Rotor Gene Q User Manual 11 2009 Analysis User Interface Remove Analysis Those channels can then be included in another allelic discrimination analysis A channel can only be used in one analysis at a time Reports Results Normalization options Discrimination Threshold This opens the Allelic Discrimination Analysis report for preview This displays the Allelic Discrimination Results window This window is opened by default when
188. t 2 Std Curves Rel Other Show Hide v Auto shrink window 2 Enter a name for the analysis Rotor Gene Q Series Software i x Enter a name for the relative quantitation analysis Cancel Relative Quant Analysis 3 Designate the pages used for normalizing gene analysis and gene of interest analysis For example clicking on Gene of Interest Standard Curve brings up the Selection of Gene of Interest Standard window Select Rotor Gene Q User Manual 11 2009 7 31 Analysis User Interface the page where the gene of interest was quantitated Repeat the procedure for the normalizing gene Optionally a calibrator can be defined If this option is selected the calibrator is assigned a value of 1 and all other sample concentrations are calculated relative to this sample Relative Quantitation x SS Reports KI Export 2 Standard Curves Relative Quantitation U Gene of Interest Standard Curve C Normaliser Standard Curve Cl Calibrator Defined IV uto shrink window Lam Selection of Gene of Interest Standar E4 Currently Selected None To select an existing analysis to use or to create a new analysis select a channel from the list then click Select This window will close and you will be taken to the main window of this analysis Cycling A Green w FAM Normalizing Gene E Cycling Yellow wf JOE Gene Of Interest Select Dther Run Ca
189. t Details about these circumstances are given in a box like this one DE CAUTION ACHTUNG ACHTUNG weist auf Situationen und Umst nde hin die zu einer Besch digung des Ger tes f hren k nnen Um einen Ger teschaden zu vermeiden muss die genannte Anleitung unbedingt befolgt werden N here Angaben zu der Art der Gef hrdung und der Vermeidung solcher Situationen werden in einem Textfeld wie diesem gemacht FR CAUTION ATTENTION Le terme CAUTION Attention est utilis pour signaler les situations susceptibles de provoquer des d t riorations de l instrument ou d autre mat riel Les d tails sur ces circonstances figurent dans un encadr semblable celui ci CAUTION Damage to the instrument C1 Avoid spilling water or chemicals onto the Rotor Gene Q A Damage caused by water or chemical spillage will void your warranty DE Besch digung des Ger tes Vermeiden Sie es Wasser oder Chemikalien auf dem Rotor Gene Q zu versch tten Durch versch ttetes Wasser oder versch ttete Chemikalien verursachte Ger tesch den sind nicht durch die Garantie abgedeckt Rotor Gene Q User Manual 11 2009 1 23 Safety Information FR D t rioration de l appareil Eviter de renverser de l eau ou des substances chimiques sur le Rotor Gene Q Tout dommage caus par de l eau ou des produits chimiques mettra fin la garantie CAUTION Damage to the instrument C2 Always use a locking ring on the rotor This stops
190. t Analysis Rotor Gene Q using the EpiTect HRM Kit available soon A a standard normalized melt curve and B a difference plot normalized to the 5096 methylated sample are shown Guidelines for successful HRM analysis The success of HRM analysis depends largely on the particular sequence under investigation Certain sequence motifs such as hairpin loops or other secondary structures localized regions of unusually high or low GC content or repeat sequences can all affect the outcome In addition use of standardized kits and optimized protocols from QIAGEN can overcome many of the potential challenges listed Some simple guidelines to help ensure success are detailed below Analyze small DNA fragments Analyze fragments no greater than about 250 bp Larger products can be analyzed successfully but usually provide lower resolution This is because for example a single base variation has a greater effect on the melting behavior of a 100 bp amplicon than on a 500 bp amplicon Ensure that PCR contains only specific product Samples contaminated with post PCR artifacts such as primer dimers or nonspecific products can make HRM results difficult to interpret Kits from QIAGEN for HRM analysis ensure maximum specificity with no need for optimization Use sufficient preamplification template Analysis of real time PCR data can be very useful when troubleshooting HRM analyses Amplification plots should have a C threshold cycle of less
191. t Dialog EN 3 Native Data Format ptions Size as Metafile z as PostScript width 333 as PDF as POX Height 1540 as GIF as PNG as JPEG v Keep aspect ratio 4 Check the Keep aspect ratio checkbox to keep the image in the correct proportion when adjusting its size 5 Click Save and select a filename and location for the file in the dialog box which appears If a higher resolution image is required we recommend either increasing the size of the image until it meets your requirements or saving the graph as a Metafile emf Rotor Gene Q User Manual 11 2009 8 3 Additional Functions wmf This is a vector based format that can be opened in software such as Adobe Illustrator allowing the user to create an image of any resolution Native format export Graphs in the Rotor Gene Q software use the third party TeeChart component developed by Steema software To save a graph in native format select the Native tab in the Export Dialog window see previous screenshot and then click Save Native format is the standard TeeChart file format This allows the user to use TeeChart Office from Steema software TeeChart Office is available as freeware and is installed as part of the Rotor Gene Q software package To access the software click on the TeeChart icon on the desktop dh TeeChart Office TeeChart Office enables manipulation of exported graphs including
192. t applications The approach used should be validated x NTE Threshold Percentage of largest Fl change 0 15x Any sample with a total change in fluorescence 2 less than this percent relative to the largest change in any tube will not be displayed Reaction Efficiency Threshold Enabled Only samples which have an individual reaction effiency at least equal to this Threshold 100 value will be displayed after ral normalization 7 26 Rotor Gene Q User Manual 11 2009 Analysis User Interface NTC Threshold This allows samples or NTCs that have a slight drift upwards to be excluded from analysis All samples with a change below the NTC Threshold will not be reported The percentage is relative to the largest maximum change found in any tube For example if one sample began at a background of 2 Fl and increased to 47 FI then 45 Fl represents 10096 An NTC Threshold of 1096 would consider any sample less than 4 5 Fl as noise Reaction The Reaction Efficiency Threshold is an Efficiency alternative method of excluding noise from Threshold analysis This normalizing algorithm uses the reaction efficiency estimation techniques used in comparative quantitation see Section 7 6 6 All samples that do not have a reaction efficiency of at least this level are excluded A level of 096 indicates that during the exponential phase no reaction took place 10096 indicates that a completely ef
193. t curve analysis analyzes the derivative of the raw data after smoothing This analysis is commonly used for genotyping and allelic discrimination Peaks in the curve are grouped into bins and all peaks below the threshold are discarded Bins can then be mapped to genotypes using the Genotypes command Rotor Gene Q User Manual 11 2009 7 37 Analysis User Interface After a run has finished for some chemistries a melt step can be added to visualize the dissociation kinetics of the amplified products The temperature is increased at a linear rate and the fluorescence of each sample is recorded A typical melt curve analysis is shown below Hf Rotor Gene Q Series Software SybrMelts 581 xl File Analysis Run Gain View Window Help A Uhd PHBH 9 e 5 s B w si oO New Open Save Start Pause Stop Help Settings Progress Profile Temp Samples Analysis Reports Arrange channels JT Melt A CHL 7 Melt A che IB ier curve a A ACHE Pose _ IDI Page Poe El amp Reports Results Genotypes Threshold Bank On Bank Off NamedOn AllOn All Off Edit Samples Peak Calling Iv Flip sign of dF dT Threshold 03089 Genotype Peak 1 Temperature SS x Heterozygous 88 5 Bin B Threshold K
194. than or equal to 30 cycles Products that amplify later than this due to low starting template amount or template degradation typically produce variable HRM results due to PCR artifacts Rotor Gene Q User Manual 11 2009 11 7 High Resolution Melt Analysis Normalize template concentration The amount of template added to the reaction should be consistent Normalize the starting concentrations so that all amplification plots are within 3 C values of each other This ensures input concentrations are within a 10 fold range Check for aberrant amplification plots Prior to running HRM examine amplification plot data carefully for abnormal amplification plot shape Plots with a log linear phase that is not steep is jagged or that reaches a low signal plateau compared to other reactions can indicate poor amplification or a fluorescence signal that is too low e g this could occur if primer concentration was too low Poor reactions can be caused by reaction inhibitors or incorrect reaction setup HRM data from such samples can be inconclusive or of low resolution To avoid unreliable results we recommend QIAGEN kits for sample preparation and HRM analysis Keep postamplification sample concentrations similar The concentration of a DNA fragment affects its melting temperature T For this reason sample DNA concentrations must be kept as similar as possible When analyzing PCR products ensure that every reaction has amplified
195. the analysis is first displayed A variety of options are available to optimize raw data normalization E Dynamic Tube dynamic tube normalization M Slope Correct noise slope correction M Ignore First x cycles correction for noise in initial cycles For more details see page 7 25 Enter values in these text boxes to position the discrimination threshold All curves passing this threshold are considered to be genotyping samples Click on the icon to the right of each text box then drag the threshold on the graph to set these values visually Discrimination Threshold Ihemod 0 05902 EE 9 K Rotor Gene Q User Manual 11 2009 7 45 Analysis User Interface 7 6 8 Genotypes This opens the Genotyping window which is used to define which genotype is detected in each channel This window enables genotypes to be assigned to channels for allelic discrimination analysis In the example below a sample is heterozygous if readings in channels Cycling A Green and Cycling A Yellow cross the threshold Ze Genotyping x Reacting Channels wild Type Cycling A Green Heterozygous Cycling A Green Cycling Yellow Mutant Cycling A Yellow zi Allelic analysis Allelic analysis templates enable export of templates normalization threshold and genotype settings into a single alt file This file can be imported and reapplied in other experiments See Section 8 1 for more details Imported Se
196. the instrument from an AC power outlet that has no ground earth connection PC requirements The laptop computer supplied with the Rotor Gene Q fulfills the requirements of the Rotor Gene Q software detailed in the following table PC system requirements Description Minimum requirement Operating system Microsoft Windows XP Processor Pentium IV or higher 2 GHz Main memory 512 MB RAM Hard disk space 10 GB HDD Rotor Gene Q User Manual 11 2009 4 1 Installation Procedures 4 2 4 2 Unpacking the Rotor Gene Q The Rotor Gene Q is delivered with all the necessary components for setting up and running the instrument The box also contains a list of all the components provided It is recommended to check this list to ensure that all the components are present The accessories box sits on top of the foam packing The accessories box contains Installation CD Loading Block 96 x 0 2 ml Tubes Loading Block 72 x 0 1 ml Tubes Rotor Holder dismantled for safe transport 36 Well Rotor this rotor is red in color 36 Well Rotor Locking Ring The following items are packed on each side of the foam packing BH USB and RS 232 serial cable M International power cable set M PCR Tubes 0 2 ml 1000 M Strip Tubes and Caps 0 1 ml 1000 Once all these components have been removed from the box remove the foam packing on top of the Rotor Gene Q Carefully remove the Rotor Gene Q from the box and unwrap the pl
197. this technique 1 To perform the analysis select Other and then Comparative quantitation in the Analysis window Double click on the channel to analyze Warton K Foster N C Gold W A and Stanley K K 2004 A novel gene family induced by acute inflammation in endothelial cells Gene 342 85 Rotor Gene Q User Manual 11 2009 7 41 Analysis User Interface 2 Choose a control sample using the drop down menu on the right hand side of the screen below the toggler 3 The results are automatically calculated and displayed in the Comparative Quantitation Results window below the graph The first columns of the Comparative Quantitation Results window show the sample number and name The Takeoff column gives the takeoff point of the sample The second derivative of the amplification plot produces peaks corresponding to the maximum rate of fluorescence increase in the reaction The takeoff point is defined as the cycle at which the second derivative is at 2096 of the maximum level and indicates the end of the noise and the transition to the exponential phase This graph shows a second derivative of an amplification plot showing the relative positions of the second derivative peak and the takeoff point Second derivative maximum The Amplification column provides the efficiency of the sample A 10096 efficient reaction would result in an amplification value of 2 for every sample which means that the ampli
198. ticular run and the reaction efficiency has not varied 7 16 Rotor Gene Q User Manual 11 2009 Analysis User Interface between 2 runs Curves can be imported from another channel or from another run by clicking on Import Curve It is possible to adjust the standard curve if required Adjusting the standard curve means that only the efficiency of the source standard curve is imported into the current run Whether or not the standard curve should be adjusted depends on the chemistry used To adjust the standard curve use a reference in the new run with a known concentration Define a reference by setting the sample type to Standard and entering a concentration value in the Edit Samples window Multiple copies of the same reference can be entered to improve accuracy Note that it is not possible to define more than one reference concentration or standard For example it is possible to have 3 replicate references of 1000 copies but it is not possible to have one reference of 1000 copies and another with 100 copies in the same run Once the standard curve has been imported the standard curve type changes to Fixed Click Reset to change the standard curve type back to Floating A screenshot of the Import Standard Curve window is shown below Import Standard Curve x Import Standard Curve Current Run C From Other Run Channels Cycling A Yelow conc 10 0 301 CT 12 525 CT 3
199. tional 4 reference wells It enables integration of the Rotor Gene Q with 96 well laboratory workflows The extra wells can be conveniently used for more samples additional control reactions or orientation reactions without occupying any of the standard 96 well positions For seamless 96 well workflow compatibility Rotor Disc 100 wells use 96 well plate labeling conventions i e A1 A12 through to H1 H12 The additional 4 reference wells are labeled R1 R4 For more information on the Rotor Disc 100 see Section 5 3 Rotor specifications Recommended Well reaction Rotor type capacity Sample no Tube type volume 36 Well Rotor 200 ul 36 PCR Tubes 20 50 ul 0 2 ml 72 Well Rotor 100 ul 72 Strip Tubes and 10 50 ul Caps 0 1 ml Rotor Disc 72 100 ul 72 Rotor Disc 72 20 25 ul Rotor Rotor Disc 100 30 ul 100 Rotor Disc 100 15 25 ul Rotor Rotor Gene Q User Manual 11 2009 5 3 Operating Procedures Hardware 5 2 5 4 Note The 36 Well Rotor and 72 Well Rotor for the Rotor Gene Q are not to be used on Rotor Gene 3000 instruments due to optical alignment incompatibilities Please continue to use the older 36 position and 72 position rotors with Rotor Gene 3000 instruments Note With 0 1 ml strip tubes volumes as small as 5 ul can be used when QIAGEN Vapor Lock PCR overlay is applied Reaction setup IMPORTANT Adequate controls should be used in each run to ensure reliable results Reactions can be prepared us
200. tioned anywhere in the rotor and can belong to multiple groups 1 To define a group type the full name of the group next to a sample and then press ENTER 7 78 Rotor Gene Q User Manual 11 2009 Analysis User Interface Edit Samples nt xl File Edit Format Security Standard Rotor Style pe Given Conc Format Unit Copies More Options r Samples Treated TL eg B ED Name Type Groups Given Conc Selected 41 Tissue Unknown Yes 42 Tissue Unknown Yes 43 Tissue Unknown Yes A4 Tissue Unknown Yes S A5 Lung Unknown Yes Ab Lung Unknown Yes s Luna Unknown ERES Yes ES AS Lung Unknown Ere Yes B1 None No B2 None No B3 None No B4 None No B5 None 2 The Edit Group window appears f Edit Group x Group Properties Code TREAT Name T reated 3 Define a suitable abbreviation then click OK The abbreviation can now be used to set up groups Aggregate results such as average value and 95 confidence intervals are calculated automatically for groups in any analysis A Name Type Ct Given Conc Cop Calc Conc Copie Var Rep Ct Rep Ct Stc Rep Ct 95 CI Rep Tissue Unknown 18 82 18 75 0 17 18 48 19 02 Tissue Unknown 18 75 Tissue Unknown 18 82 Tissue Unknown 18 52 Lung Unknown 18 73 18 70 0 09 18 55 18 85 Lung Unknown 18 62 Lung Unknown 18 81 Lung Unknown 18 63 Treated Group 18 72 0 13 18 52 18 83
201. to open an existing sample template or to save sample names as a template for future use The extension of these template files is smp The Edit menu allows rows to be copied and pasted The Security menu enables the sample definitions to be locked 7 70 Rotor Gene Q User Manual 11 2009 Analysis User Interface Given Conc Format Unit Line style Edit Reset Default Gradient Ll icon Samples u H E dit Reset Default Gradient This drop down menu is used to choose a suitable format for the concentration display Concentrations are automatically formatted according to the currently selected location This drop down menu sets the units of measurement for the assay The style of the line can be modified to improve readability of graphs on black and white printers Certain lines can be emphasized by modifying their style To access this feature click on the right arrow button next to the Edit button Pressing Edit opens the color selector Multiple rows can be selected when assigning a color to tubes Click Reset Default to reset all selected color cells back to their default color values Gradient allows a gradient to be chosen from the first to the last selected color Several gradients can be defined in a sample setup The New icon clears the Edit Samples window in preparation for data entry Rotor Gene Q User Manual 11 2009 7 71 Analysis User Interf
202. to the plateau phase At plateau all reactions will have amplified to a similar extent irrespective of their starting amount Note however that poor reactions may not reach plateau with the same amplified quantity due for example to inconsistent assay setup e g the primer concentration was too low Ensure sample to sample uniformity All samples must be of equal volume and should contain the same concentration of dye DNA melting behavior is affected by salts in the reaction mix so it is important that the concentration of buffer Mg and other salts are as uniform as possible in all samples Similarly use only identical reaction tubes from the same manufacturer to avoid 11 8 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis variations due to plastic thickness and autofluorescence properties Allow sufficient data collection for premelt and postmelt phases Capture HRM data points over approximately a 10 C range centered around the observed T see figure on page 11 1 This provides sufficient baseline data points for effective curve normalization and will result in more reproducible replicates and easier data interpretation 11 6 Sample preparation Sample degradation should be avoided during purification and storage Avoid excessive amounts of inhibitors such as from ethanol carryover To improve HRM results we recommended keeping the amount of template used consistent between samples Spectrophotome
203. tor The Rotor Gene Q User Manual 11 2009 Operating Procedures Software reference tube must also contain a detection chemistry that enables detection of strand dissociation When heating to the initial denature temperature a melt is performed on the green channel from 80 C to 95 C by default The parameters of this initial melt can be adjusted by the user From this data a melt curve is generated and automatically analyzed 75 80 85 90 95 10C Se The melt peak is referenced back to the raw data to obtain a denature threshold Then every Optical Denature Cycling step the instrument is heated as quickly as possible and data is acquired continually Once the reference tube has reached the denature threshold fluorescence level the instrument is immediately cooled and proceeds to the next programmed step in the cycle A peak is not calculated while cycling Instead the fluorescence level is referenced to the melt peak and this designates the denature threshold In the following graph the raw fluorescence readings and the first derivative have been overlaid This shows the correspondence between the denature threshold and the melt peak obtained during the calibration Rotor Gene Q User Manual 11 2009 6 19 Operating Procedures Software 6 20 1 3 M ns 1 gu n N f N ae Denature nn threshold 05 0 25 D p 75 80 85 80 c To perform Optical Denature Cycling you will need MB A
204. tric analysis for determining DNA concentration and purity is recommended We recommend QIAGEN kits for sample preparation Note At 260 nm one absorbance unit is equal to 50 ug ml DNA Pure DNA will provide a 260 nm to 280 nm ratio of 1 8 11 7 Software setup 1 Open a new run file by selecting New from the File menu In the Advanced wizard select HRM Rotor Gene Q User Manual 11 2009 11 9 High Resolution Melt Analysis x Quick Start Advanced Corbett Life Science High Resolution Melt run HRM with Pre Amplification New Cancel Help M Show This Screen When Software Opens 2 Set the rotor type in this example the 72 Well Rotor is used Ensure that the locking ring is in place and the Locking Ring Attached checkbox is checked before proceeding to the next step 11 10 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis New Run Wizard x Welcome to the Advanced Run Wizard r Rotor Type 36 Well Rotor Rotor Disc 72 Rotor Disc 100 IV Locking Ring Attached Skip Wizard lt lt Back Next gt gt 3 Setthe run details Enter the operator name optional and add any notes about the experiment optional Select the reaction volume required and sample layout desired x This screen displays miscellaneous options for the run Complete the fields Mis ee in Clicking Next when you are ready to move to the next page the wiz
205. ttings lt none gt Import Export Scatter graph analysis Scatter graph analysis enables genotyping based on the relative expression of amplification plots across 2 channels Unlike allelic discrimination genotype is decided based on regions defined from the scatter graph rather than from a 7 46 Rotor Gene Q User Manual 11 2009 Analysis User Interface single threshold To perform this analysis select Other and then Scatter Graph Analysis in the Analysis window When performing scatter graph analysis it is not sufficient to double click on one channel to analyze because this analysis is performed using 2 channels simultaneously To perform this analysis either hold down SHIFT and click to highlight the channels to analyze or drag the mouse pointer over the channels Once the desired channels have been highlighted click Show The list will update to show all the channels on one line with a checkmark next to them This indicates that they will all be used in one analysis To remove one or more of these channels right click on the analysis and select Remove Analysis Those channels can then be included in another scatter graph analysis A channel can only be used in one analysis at a time Reports This opens the Scatter Analysis report for preview Results This displays the Scatter Analysis Results window The genotype for each sample is determined by the regions defined by the user
206. tus lights 3 1 am General Description General Description The Rotor Gene Q is an innovative instrument that enables high precision real time PCR end point PCR and high resolution melt HRM analysis It is highly suited for use in gene expression analysis genotyping pathogen detection and many other areas of research The powerful and user friendly software provides simplicity for beginners as well as an open experimental platform for advanced users Thermal performance The Rotor Gene Q uses a sophisticated heating and cooling design to achieve optimal reaction conditions The unique rotary format ensures optimal thermal and optical uniformity between samples which is critical for precise and reliable analysis Samples spin continually at 400 rpm during a run Centrifugation prevents condensation and removes air Rotor Gene Q User Manual 11 2009 3 1 General Description bubbles but does not pellet DNA In addition samples do not need to be spun down prior to a run Samples are heated and cooled in a low mass air oven Heating is achieved by a nickel chrome element in the lid The chamber is cooled by venting the air out through the top of the chamber while ambient air is blown up through the base Heating e emn Heater Spin uga e dnves elements air around chamber switch on Chamber vent Seals to contain air Cooling Heater elem ent S Switch off Centrifugal fan
207. u have insufficent rights to use the software Please contact the domain administrator to set up groups 7 9 2 Running multiple users on the same computer To use the Rotor Gene Q software with multiple users create a user account that does not have access to the Rotor Gene Q software Log into windows using this account so that users cannot anonymously access the Rotor Gene Q 1 Using the Rotor Gene Q Software Login icon users can open their user account in the Rotor Gene Q software Rotor Gene Q User Manual 11 2009 7 91 Analysis User Interface Ze Rotor Gene Q Software Login 2 Enter the username and password in the box that appears x Run Rotor Gene As Other User Domain CORIT198 Username linsonay Cancel Password 3 The domain is either the computer you are logging into or the name of your local network Consult your network administrator if unsure which domain to enter in this field Note After logging in all of the user files will be available for that user Each user can save files in their own area This ensures a high level of security Note Each user should log out after their run has been completed to prevent other users from performing a run in their name 7 9 3 Audit trails Every time a file is saved by a user their details are recorded in Run Settings under the Messages tab as Security Audit Trail Summary and Security Audit Trail Detail 7 92 Roto
208. uch as the unique identifier of the user This identifier is important to avoid a user creating an account with the same name on another computer and thereby impersonating another user In this case the user names will be the same but the account IDs will be different Rotor Gene Q User Manual 11 2009 7 93 Analysis User Interface 7 9 4 The identifier for the account CORITOO1 RGQ User 2 S 1 5 21 1177238915 195 is shown in the details BERrstor Gene Q Series Software VIRTUAL MODE Three Step with Melt 2008 11 26 1 8 xl File Run Gain View Window Help AOU PHB Ol f B Ww Oo Settings Progress Profile Temp Samples Arrange New Open Save Start Pause Stop ox Run Settings General Machine Options Messages Channels Tube Layout Security Run Wizard Advanced E Security Audit Trail Summary Saved At 26 11 2008 2 47 45 PM By CORITOD1 RGQ User 2 Experiment Started At 26 11 2008 2 47 46 PM By CORITODT RGO User 2 Saved At 26 11 2008 3 36 00 PM By CORITOOT RGQ User 2 Saved At 26 11 2008 3 36 12 PM By CORITOO1 RGQ User 2 Saved At 26 11 2008 3 41 38 PM By CORITOD1 RGQ User 2 E Security Audit Trail Detail 00 52 00 53 Saved At 26 11 2008 2 47 45 PM By CORITOD1 RGQ User 2 5 1 5 21 1177238915 19 5 Experiment Started At 26 11 2008 2 47 46 PM By CORITODI RGQ User 2 5 1 5 21 117 Saved At 26 11 2008 3 36 00 PM By CORITODT RGQ User 2 5 1 5 21 1177238915 19 Close Saved At 2 amp
209. uding all relevant information from a run The Save As option will save all the information to a file that you can copy onto a disk or across a network if you do not have access to e mail on the computer running the Rotor Gene Q Rotor Gene Q User Manual 11 2009 8 2 8 3 Additional Functions Additional Functions Analysis templates Some analyses require the user to define thresholds normalization settings and genotype settings Often these settings are reused frequently in multiple experiments Analysis templates enable the user to save and reuse these settings This reduces the effort of reentering settings and reduces the risk of error Quantitation Melt Allelic discrimination Scatter graph analysis and EndPoint analysis support analysis templates These analyses allow the user to export a template that is unique to the analysis e g Quantitation analysis allows export and import of qut files that contain quantitation settings After an analysis template has been imported or exported the filename of the template is displayed for future reference Imported Settings ANTemplatessBC qut Import Export Opening a second run While performing a run it is possible to open and analyze runs that were performed earlier Several functions such as New or Start Run buttons are not activated in the second window A new run can be started from the first window once the first run has finished
210. uired due to incorrect maintenance WARNING Risk of personal injury and material damage W2 Rotor Gene Q is a heavy instrument To avoid personal A injury or damage to the instrument take care when lifting WARNING Risk of personal injury and material damage W3 Do not attempt to move the Rotor Gene Q during A operation CAUTION Damage to the instrument C1 Avoid spilling water or chemicals onto the Rotor Gene Q A Damage caused by water or chemical spillage will void your warranty In case of emergency switch off the Rotor Gene Q at the power switch at the back of the instrument and unplug the power cord from the power outlet WARNING Risk of personal injury and material damage WA Do not try to open the lid during an experiment or while A the Rotor Gene Q is spinning Otherwise if you overcome the lid lock and reach inside you risk contact with parts that are hot electrically live or moving at high speed and you may injure yourself and damage the instrument 1 2 Rotor Gene Q User Manual 11 2009 WARNING gt Safety Information Risk of personal injury and material damage W5 If you need to stop an experiment quickly turn off the power to the instrument then open the lid Let the chamber cool before reaching inside Otherwise you risk injury by touching parts that are hot WARNING gt Risk of personal injury and material damage w If the equipment is used i
211. ument maintenance and then OTV The wizard prompts for the OTV serial number This number Rotor Gene Q User Manual 11 2009 Optical Temperature Verification can be read from the label on the OTV Rotor Disc or can be imported from the CD by clicking on Browse and choosing the otv file provided on the CD Once the number is entered click Start x Quick Start Advanced KI Perform Last Run S Empty Run 3 Three Step with Melt e e Two Step i HRM d Other Runs Instrument Maintenance Open A Template In Another Folder New Cancel di Help M Show This Screen When Software Opens Rotor Gene Q User Manual 11 2009 10 3 Optical Temperature Verification D Optical Temperature Verification Rotor se This template allows you to perform in tube temperature verification with the QIAGEN gt OTY Rotor For more information see our website available from the Help menu Rotor Details Fluorescent insert s placed correctly Please enter the serial number of the rotor you are using OTV C172 31F4 2D0 384 306 3B 30 i Temperatures 50 72 90 Expiry Date Mar 2006 Validity Check Valid Start Exit 4 The software then prompts for a filename for the run Then the run begins 5 The run performs a series of melts that determine the thermal characteristics of the Rotor Gene Q Pi OTY Temperature Verification Results x Summar
212. w orange and red channels are standard configurations for 4 channel multiplex detection Tube Layout If using a 72 Well Rotor samples can be arranged to closely match the labeling on a 9 x 8 block By default the tube layout tab allows samples to be labeled sequentially i e 1 2 3 This means that samples are labeled consecutively in the order in which they are placed in the Rotor Gene Q Alternatively samples can be labeled 1A 1B 1C etc This option can be useful if samples were set up with a multichannel pipet Security The security tab displays information about the run signature The run signature is an irreversible key that is regenerated every time the file is changed If any section of the rex file is modified outside the software the signature and the file will no longer match Checking the signature allows confirmation that the raw data was not modified outside the application that the profile has not been tampered with and that the temperature graph is valid The signature also protects against corruption such as file system errors Note If rex files are e mailed the encryption process can invalidate the signature To avoid this zip the file before e mailing Rotor Gene Q User Manual 11 2009 7 67 Analysis User Interface 7 8 2 Run Settings xf r General Information E Run Signatures are stored within all newly saved runs These signatures like a wax General Machine Options
213. x Reading Min Gain Max Gain Edit Green 1 1FI FFI 10 10 Remove Aid Remove All RR T NI Manual Close Help Ko 10 Start the run by clicking Start Run and save the run file to your computer New Run Wizard Summary Green Gain 7 Rotor 72 Well Rotor Sample Layout er d te Reaction Volume in microliters 25 Start Run Once you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template to save settings for future runs 11 16 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis 11 Edit the sample names optional Sample names can be edited during or after a run BER File Edit Format Security Standard Rotor Style m Settings Given Conc Format DI Unit copies ul DI More ptions p ee ie 0 mr 2 wild type 1 Unknown x E 3 wild type 1 Unknown No 4 mutation 1 Unknown No 5 mutation 1 Unknown No B mutation 1 Unknown No 7 heterozygote 1 Unknown No 8 heterozygote 1 Unknown No 9 heterozygote 1 Unknown No 10 Unknown No 11 Unknown No 12 Unknown No m Page Name Page 1 ES pz New Delete Synchronize pages Undo DK Cancel Help 11 8 Real time PCR data analysis Analysis of the real time PCR data prior to HRM data analysis is advantageous Real time PCR data can highlight poorly performing assays Identifying these outl
214. y Verification Result Adjustment Recommended Details Lower Point Mo Adjustment Required Middle Point No Adjustment Required Upper Point Adjustment Required Apply Adjustment 6 When the run is finished the software indicates whether the Rotor Gene Q is within specification 7 If adjustment is required the user should click Apply Adjustment This prompts the user to perform a verification run After the verification run is complete no 10 4 Rotor Gene Q User Manual 11 2009 Optical Temperature Verification adjustment should be required If further adjustment is required contact your distributor 8 When the Rotor Gene Q is within specification a report of the run can be reviewed and printed Rotor Gene Q User Manual 11 2009 10 5 Optical Temperature Verification This page intentionally left blank 10 6 Rotor Gene Q User Manual 11 2009 High Resolution Melt Analysis 11 High Resolution Melt Analysis High resolution melt HRM analysis is an innovative technique that is based on analysis of DNA melting HRM characterizes DNA samples according to their dissociation behavior as they transition from double stranded DNA dsDNA to single stranded DNA ssDNA with increasing temperature see Figure below A HRM instrument collects fluorescent signals with extremely high optical and thermal precision creating many application possibilities Melt curve Premelt ph
215. ysis Quantitation Concise Quantitation Full Report ee Quantitation Standard Report SR Setup The initial setup of the Rotor Gene Q should be completed during installation However this option allows you to change the Rotor Gene Q connection setup if you wish to do so after installation Rotor Gene Q User Manual 11 2009 7 9 Analysis User Interface SS E General Miscellaneous Options The software can simulate basic operation of the machine for testing and training purposes Ticking this box will enable a Virtual Machine simulation Virtual Mode Untick this box to prevent further access to this setup Screen v Allow access to this setup screen Fort COM e amp uto Detect Virtual Mode Allow access to this setup screen Port Select this option if the software will be used without a connected Rotor Gene Q The software retains all functions This mode is useful for demonstration purposes data analysis and setting up templates If this option is not checked during setup this window can no longer be accessed This security measure prevents users from altering the settings To reestablish access contact your distributor Select the correct communication port to enable communications between the computer and the Rotor Gene Q If you are unsure which port to select click Auto Detect to search for all available ports 7 10

Rotor-Gene Q User Manual - Faculté de médecine de l`université d

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Rotor-Gene Q User Manual - Facult&eacute; de m&eacute;decine de l`universit&eacute; d

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Rotor-Gene Q User Manual - Faculté de médecine de l`université d