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 Rotor-Gene Q MDx CE User Manual    02/2022 

103 

 

6.6.5

 

Melt curve analysis 

Melt 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. 

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. 

 

Содержание Rotor-Gene Q MDx CE

Страница 1: ...Sample to Insight__ February 2022 Rotor Gene Q MDx CE User Manual 9002022 9002032 9002042 QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden GERMANY R1...

Страница 2: ...ut not provided 11 2 Safety Information 12 2 1 Proper use 13 2 2 Electrical safety 15 2 3 Biological safety 16 2 4 Chemical safety 17 2 5 Waste disposal 18 2 6 Mechanical hazards 18 2 7 Maintenance sa...

Страница 3: ...MDx instruments 32 4 2 Site requirements 39 4 3 AC power connection 40 4 3 1 Power requirements 40 4 3 2 Grounding requirements 40 4 3 3 Installation of AC power cord 40 4 4 Configuration for Windows...

Страница 4: ...enu 83 6 6 1 Analysis 83 6 6 2 Quantitation 85 6 6 3 Two standard curve 96 6 6 4 Delta delta CT relative quantitation 100 6 6 5 Melt curve analysis 103 6 6 6 Comparative quantitation 106 6 6 7 Allelic...

Страница 5: ...es 147 6 10 Gain menu 147 6 11 Window menu 148 6 12 Help function 148 6 12 1 Send Support E Mail 149 7 Additional Functions 153 7 1 Analysis templates 153 7 2 Opening a second run 153 7 3 Scaling opti...

Страница 6: ...3 Error and warning messages 185 11 3 1 General instrument errors 185 11 3 2 Rotor Gene Q Software messages 187 12 Glossary 190 13 Technical Specifications 191 13 1 Environmental conditions operating...

Страница 7: ...ix B Mathematical Techniques 201 15 1 Quantitation 201 15 1 1 Confidence intervals for calculated concentrations 201 15 1 2 Confidence intervals for CT values 202 16 Ordering Information 203 16 1 Roto...

Страница 8: ...ure safe operation of the instrument and to maintain the instrument in a safe condition Note that the Rotor Gene Q MDx comes in multiple configurations For details including ordering information pleas...

Страница 9: ...suggestions about product performance or new applications and techniques For technical assistance contact QIAGEN Technical Services For up to date information about the Rotor Gene Q MDx visit https w...

Страница 10: ...e Rotor Gene Q MDx 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 MDx instrume...

Страница 11: ...o 9026760 72 Well Rotor cat no 9018903 Locking Ring 72 Well Rotor cat no 9018904 Loading Block 72 x 0 1 ml Tubes cat no 9018901 Rotor Holder cat no 9018908 Strip Tubes and Caps 0 1 ml 250 cat no 98110...

Страница 12: ...could result in personal injury to you or others 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...

Страница 13: ...incorrect maintenance WARNING Risk of personal injury and material damage The Rotor Gene Q MDx is too heavy to be lifted by one person To avoid personal injury or damage to the instrument do not lift...

Страница 14: ...t then open the lid Let the chamber cool before reaching inside Otherwise you risk injury by touching parts that are hot CAUTION Risk of personal injury and material damage If the equipment is used in...

Страница 15: ...rminal 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...

Страница 16: ...upply as well as fuse ratings The equipment should only be operated under these conditions 2 3 Biological safety Specimens and reagents containing materials from biological sources should be treated a...

Страница 17: ...ACGIH American Conference of Government Industrial Hygienists United States of America COSHH Control of Substances Hazardous to Health United Kingdom 2 4 Chemical safety WARNING Hazardous chemicals S...

Страница 18: ...quipment WEEE page 196 2 6 Mechanical hazards The lid of the Rotor Gene Q MDx must remain closed during operation of the instrument WARNING Moving parts To avoid contact with moving parts during opera...

Страница 19: ...0 min before attempting to manually open the lid WARNING Risk of overheating To ensure proper ventilation maintain a minimum clearance of 10 cm at the sides and rear of the Rotor Gene Q MDx Slits and...

Страница 20: ...cifically described in this user manual WARNING Risk of fire When cleaning the Rotor Gene Q MDx with alcohol based disinfectant leave the Rotor Gene Q MDx door open to allow flammable vapors to disper...

Страница 21: ...of the instrument In vitro diagnostic medical device Type plate on the back of the instrument CSA listing mark for Canada and the USA Type plate on the right side panel Legal manufacturer Type plate o...

Страница 22: ...MDx 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...

Страница 23: ...ce 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 t...

Страница 24: ...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...

Страница 25: ...nd the vents can impact the performance of the instrument 3 2 2 Lid handle The lid handle is used to slide the lid of the instrument back this handle is not intended to support the weight of the instr...

Страница 26: ...s of the Rotor Gene Q MDx Internal view of the Rotor Gene Q chamber 1 Rotor hub 2 Optical lens 3 3 1 Rotor hub The rotor hub holds the rotor in place within the instrument 3 3 2 Optical lens Optical l...

Страница 27: ...and running the instrument The box also contains a list of all the components provided Note Check this list for completeness to ensure that all the components are present Note Check that the instrumen...

Страница 28: ...MDx has been unpacked proceed with installation as described below CAUTION Damage to the instrument When the Rotor Gene Q MDx is started immediately after delivery in cold climates mechanical parts c...

Страница 29: ...software download the software from QIAGEN com and transfer to the computer on a virus free removable media or insert the Removable Media software delivered with the instrument into the computer 2 If...

Страница 30: ...ide to the I position A blue Standby light on the front of the Rotor Gene Q MDx indicates that the instrument is ready for use Note When starting connected to a computer for the first time the Rotor G...

Страница 31: ...n list Run in Virtual Mode for demonstration Checking this box allows installation of the Rotor Gene Q software on a computer that is not connected to a Rotor Gene Q MDx The software is fully function...

Страница 32: ...ocesses during the PCR run and the data acquisition process For this reason it is important to ensure that no other processes use significant system resources and thus slow down the Rotor Gene Q softw...

Страница 33: ...s Plus V16 0 5 If the computer is used in an environment without network access please also make sure the antivirus software supports offline updates To get consistent results after installation of an...

Страница 34: ...g connections are allowed because they may be required for retrieving updates If your configuration is different QIAGEN recommends configuring the firewall in the same way as described above To this e...

Страница 35: ...sons cable based network access instead of Wi Fi shall be used The laptop computers provided by QIAGEN have a disabled Wi Fi adapter If your configuration is different a system administrator must disa...

Страница 36: ...er Manual 02 2022 36 2 Select Change adapter settings 3 Hover over Wireless Network Connection press the right mouse button and select Disable from the context menu 4 Check that the Wireless Network C...

Страница 37: ...ent tools Please be aware that due to the dynamic nature of the IT world this list may not be complete and tools may be released that are not known at the time of writing It is important that system a...

Страница 38: ...Rotor Gene Q MDx CE User Manual 02 2022 38 2 Select Change settings 3 Select Never check for updates 4 Check that the Important updates option Never check for updates is active...

Страница 39: ...not subject to large temperature fluctuations Refer to Appendix A for the weight and dimensions of Rotor Gene Q MDx instruments Ensure that the workbench is dry clean and has additional space for acc...

Страница 40: ...wer cord to the socket located at the rear of the Rotor Gene Q MDx instrument and the other end to the AC power outlet 4 4 Configuration for Windows security The laptop computers that are provided by...

Страница 41: ...set up an additional standard Windows user account to prevent access to critical system areas such as Program Files Windows directory e g access to installation or uninstallation functionality includ...

Страница 42: ...ad or mouse or equivalent is required Bluetooth Must be switched off PDF viewer or similar Must be installed not part of the software installation packages Power options Never turn off hard disks hibe...

Страница 43: ...nd RS 232 serial cable International power cable set PCR Tubes 0 2 ml 1000 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 t...

Страница 44: ...original packaging materials must be used If the original packaging materials are not applicable contact QIAGEN Technical Services Make sure that the instrument has been properly prepared see Mainten...

Страница 45: ...he 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 Optimizati...

Страница 46: ...nneal step Nucleic Acid Concentration Measurement This is a default template for measuring nucleic acid concentration using intercalating dyes HRM This folder contains high resolution melt profiles Ot...

Страница 47: ...n In the next window select the rotor type from the list Check the Locking Ring Attached checkbox and then click Next Confirm profile The cycling conditions and acquisition channels of the template ch...

Страница 48: ...window appears The run can be saved in the user s desired location 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...

Страница 49: ...o transposed letters within the sample names Therefore it is recommended to avoid using a bar code scanner and if applicable enter the sample names after the run has finished For information about set...

Страница 50: ...ling profile with data acquisition on the green channel only to speed up the run HRM This folder contains 2 high resolution melt profiles Other Runs This folder contains additional profiles Instrument...

Страница 51: ...ser 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...

Страница 52: ...nel 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 Edit Profile Aft...

Страница 53: ...he 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 cycl...

Страница 54: ...le is displayed graphically as shown in the screenshot hereafter 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 durat...

Страница 55: ...ified 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...

Страница 56: ...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 The Dye Channel Selection Chart he...

Страница 57: ...haracterizes 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 applicati...

Страница 58: ...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 coole...

Страница 59: ...elt curve on the green channel 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 ap...

Страница 60: ...ot burn the sample yet will be high enough to allow it to melt o The hold time is sufficient to denature the sample o The denature offset is set appropriately The default of 0o C is appropriate for mo...

Страница 61: ...d select Optical Denature The temperature and hold time are removed and the Optical Denature icon is displayed Gain Optimisation When setting up a new run it is helpful to use the Gain Optimisation fu...

Страница 62: ...ion 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...

Страница 63: ...p 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...

Страница 64: ...rs where the gain has been changed The cycles before the change are excluded from the analysis Note Gain Optimisation may choose a setting which does not fall within the specified range This can be du...

Страница 65: ...Start The run begins The Rotor Gene Q MDx temperature is adjusted to the temperature specified in the window The graphs in the window start to display data 3 Wait for the temperature to stabilize 4 N...

Страница 66: ...onality of this window is identical to the Edit Samples window page 127 Sample information may also be entered after the run has finished The Finish and Lock Samples button closes the screen and preve...

Страница 67: ...commend use of tubes from QIAGEN which are specially designed for use with the Rotor Gene Q MDx see Ordering Information Tubes from alternative manufacturers may autofluorescence which could affect th...

Страница 68: ...Ring enable the use of 0 2 ml tubes The tubes do not need to have optically clear caps because the Rotor Gene Q MDx reads fluorescence from the bottom of the tube rather than from the top Domed cappe...

Страница 69: ...n color The Rotor Disc 100 Rotor and Rotor Disc 100 Locking Ring enable use of 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 e...

Страница 70: ...sure reliable results Reactions can be prepared using 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...

Страница 71: ...tight seal 3 Insert the Strip Tubes into the 72 Well Rotor ensuring that each tube sits correctly in place in the correct orientation Samples will not be optimally aligned over the detection system i...

Страница 72: ...g 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 4 Insert the 72 Well Rotor Locking Ring onto th...

Страница 73: ...t Sealer The film prevents contamination by providing a strong durable and tamper proof seal Heat sealing the Rotor Disc is performed as described below Important Please read the Product Sheet supplie...

Страница 74: ...Film by slightly folding the film in half pinching the center piece and carefully tearing it out 6 Place the film over the Rotor Disc in the correct orientation as shown by the SIDE UP label Ensure t...

Страница 75: ...he Heat Sealer then push back the black catch 9 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...

Страница 76: ...change to an intermittent sound 11 Slide the Rotor Disc Loading Block out of the Rotor Disc Heat Sealer Allow the film to cool for approximately 10 s Remove the excess sealing film by pushing it down...

Страница 77: ...arrow next to the Arrange button 6 2 Toolbar These buttons are shortcuts to frequently used operations These operations can also be accessed from the drop down menus 6 3 View raw channels Click on th...

Страница 78: ...dit 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...

Страница 79: ...an run two standard curves in the green channel and generate independent reports More information on setting up sample pages is available in Section 6 8 4 Toggle Sample ID Display If a 72 Well Rotor i...

Страница 80: ...s selected the wizard guides you through the run setup and allows modification of settings and profiles For information about the templates provided see Section 5 1 1 and Section 5 1 2 New Run New Thi...

Страница 81: ...orrupted by e mail clients LIMS Export This saves the analysis in LIMS compatible formats according to the requirements of the user Please contact QIAGEN Technical Services for more information Excel...

Страница 82: ...ys the exported raw data 4 Start up the LinReg tool The tool asks you to select the cell range where the raw data are located The tool can only analyze one raw channel at a time so an appropriate regi...

Страница 83: ...no longer be accessed This security measure prevents users from altering the settings To reestablish access contact your distributor Port Select the correct communication port to enable communication...

Страница 84: ...ndow enlarges the window again Organizing the workspace Each time a new analysis is started its windows are arranged to fit in with those already on the screen If many windows are displayed this can b...

Страница 85: ...There are 3 options standard report full report and concise report Double click on the desired option to open the report in the Preview window After the report has been generated the buttons on the t...

Страница 86: ...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 root of correlation coeffi...

Страница 87: ...e 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 referenc...

Страница 88: ...old line and calculating the intersection with each of the curves the CT value for each sample is established Threshold To set the threshold click on the icon a grid with a red arrow then click and ho...

Страница 89: ...g flags are possible NEG Multi Ct The threshold crosses the fluorescence curve at least two times double intersect An unambiguous CT value cannot be determined NEG NTC The overall fluorescence increas...

Страница 90: ...f 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 di...

Страница 91: ...ents a 7 54 variation 278 974 322 611 300 000 1 07537 00 000 1 07537 Reporting an absolute value does not make sense for a standard curve The value could be reported at the lowest concentration to cre...

Страница 92: ...roduces 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 C...

Страница 93: ...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 most applications The a...

Страница 94: ...luorescence whereas setting below 0 will display samples that decreased in fluorescence during the exponential phase which should clearly be excluded Note If a value is excluded due to activation of e...

Страница 95: ...able measurement than the gradient 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 Line...

Страница 96: ...mple setup see Section Sample setup 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 normal...

Страница 97: ...interest analysis For example clicking on Gene of Interest Standard Curve brings up the Selection of Gene of Interest Standard window Select the page where the gene of interest was quantitated Repeat...

Страница 98: ...ports 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 resul...

Страница 99: ...022 99 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...

Страница 100: ...ed 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 a...

Страница 101: ...CE User Manual 02 2022 101 2 Enter a name for the analysis 3 Validation Run Performed must be checked to proceed with the analysis Define the pages where the gene of interest and normalizing gene hav...

Страница 102: ...example of results from this analysis is shown below The CT values for the gene of interest GOI CT CT values for the normalizing gene Norm CT Delta CT Delta Delta CT and relative concentration Relativ...

Страница 103: ...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 After a run has finished for some chemistries a mel...

Страница 104: ...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 cli...

Страница 105: ...ld 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 s...

Страница 106: ...ed 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...

Страница 107: ...he sign The confidence interval for a large sample number N gives a 68 3 probability that the true amplification of the samples lies within this range 1 standard deviation By doubling the interval a 9...

Страница 108: ...sult is displayed in scientific notation in the Rep Conc column of the Comparative Quantitation Results window 6 6 7 Allelic discrimination Allelic discrimination uses real time kinetic data from 2 or...

Страница 109: ...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...

Страница 110: ...e analysis is first displayed Normalization options A variety of options are available to optimize raw data normalization Dynamic Tube dynamic tube normalization Slope Correct noise slope correction I...

Страница 111: ...ied and unamplified samples at the end of a run Results are qualitative positive negative not quantitative EndPoint analysis is shown in the screenshot below EndPoint analysis is similar to allelic di...

Страница 112: ...justed by the user for each run Signal level A percentage of fluorescent signal normalized so that the highest signal of the positive controls is 100 and the lowest signal of the negative controls is...

Страница 113: ...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 cha...

Страница 114: ...al levels to within the range of 0 100 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 positi...

Страница 115: ...itive control was slightly lower Sample 4 the lowest negative control was set to 0 It is now clear that sample 5 has probably amplified whereas sample 6 has probably not amplified Note Depending on th...

Страница 116: ...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 still be defined as a positive control Since the highest positi...

Страница 117: ...bove a sample is heterozygous if readings in channels Cycling A Green and Cycling A Yellow cross the threshold EndPoint analysis templates EndPoint analysis templates allow the user to export genotype...

Страница 118: ...is 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 l...

Страница 119: ...ch run Analysis Concentration analysis operates by relating the fluorescence level to a concentration value Two analysis models are available The optimal analysis to choose depends on the chemistry an...

Страница 120: ...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 perf...

Страница 121: ...th the gain settings for each channel 6 7 2 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...

Страница 122: ...s the setup of run information run filename analysis date operator and any associated notes The window contains all information except for the profile required to configure a run After a run has finis...

Страница 123: ...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 ru...

Страница 124: ...detection filter The filters can be chosen using the drop down menu next to each window Channels Green yellow orange and red channels are standard configurations for 4 channel multiplex detection Tube...

Страница 125: ...uch 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 6 8 2 Temperature Graph Select Temperature Grap...

Страница 126: ...w 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 i...

Страница 127: ...isting 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...

Страница 128: ...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 cu...

Страница 129: ...wing the raw data the sample definitions used to display the data can be selected using the drop down menu next to the Options button The sample page to use when performing an analysis can be selected...

Страница 130: ...sample file smp and loaded into future runs with the same sample configuration Rotor Style This tab in the Edit Samples window provides an alternative way of entering sample names Select replicates by...

Страница 131: ...age suitability reduces the number of analysis options available to just include those relevant for the particular assay The Sample Page Suitability window is shown below Note When setting up an assay...

Страница 132: ...efine 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 group...

Страница 133: ...ave previously been set not to display again This may be useful for a new user who is unfamiliar with the Rotor Gene Q MDx or the Rotor Gene Q software 6 9 Access Protection for Rotor Gene Q Software...

Страница 134: ...a Linux Samba domain You must have either a local logon or a Windows server to use the security features 6 9 1 Configuration for Windows 7 This section describes how to set up the system to run Rotor...

Страница 135: ...Groups on the left side 2 In the window that appears select the Users folder Right click on the right hand window and select New User 3 Enter a username and password By default the user will be create...

Страница 136: ...n to Windows as an administrator or use the Rotor Gene Q Software Login icon to open the software and log in Note To create the RG Groups with the Rotor Gene Q software it is necessary to run the soft...

Страница 137: ...assign the user linsongy to the RG Analyst group by selecting the group on the left hand side then clicking the button Groups can be removed by selecting them then clicking the button 6 Now log in as...

Страница 138: ...oved again Then the software needs to be launched again This time the Analysis menu and Reports button are missing and the Run menu is enabled The status bar indicates that the user linsongy belongs t...

Страница 139: ...revents 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...

Страница 140: ...ports 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 a...

Страница 141: ...ser account in the Rotor Gene Q software 2 Enter the username and password mandatory in the box that appears 3 The domain is either the computer you are logging into or the name of your local network...

Страница 142: ...y Audit Trail Detail This can be used to monitor who has modified the contents of a file The Security Audit Trail Detail contains more detail such as the unique identifier of the user This identifier...

Страница 143: ...Rotor Gene Q MDx CE User Manual 02 2022 143 The identifier for the account CORIT001 RGQ User 2 S 1 5 21 1177238915 195 is shown in the details...

Страница 144: ...a shared area Run Signatures provide extra security The screenshot shows the Security tab in Run Settings for a file with a Run Signature The Run Signature is a long word that is generated each time t...

Страница 145: ...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 securel...

Страница 146: ...ll appear Click Yes to confirm Once the samples are locked it will not be possible to edit samples in the Edit Samples window Samples can also be locked and unlocked in the Edit Samples window However...

Страница 147: ...mplate 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 wi...

Страница 148: ...This accesses the Help function Virtual Demo This links to a QIAGEN website page with an interactive demonstration of the software Software Version History This gives a brief overview of new features...

Страница 149: ...work if you do not have access to e mail on the computer running the Rotor Gene Q MDx If you use the support e mail function on the laptop computer provided optionally with the Rotor Gene Q MDx countr...

Страница 150: ...in your e mail address and press Next The E mail Server Names window will open 3 Select the type of mail server for incoming mails and specify the server names for incoming and outgoing e mails Then p...

Страница 151: ...uses secure password authentication Then click Next The Congratulations window will open 5 Confirm with Finish to complete the e mail account setup Setting up in Outlook 1 Open Outlook Express from th...

Страница 152: ...mportant To avoid any retrieval of e mails during PCR runs disable the default entries in the Send Receive Messages screen 3 Disable Send and receive messages at startup 4 Disable Check for new messag...

Страница 153: ...t 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...

Страница 154: ...e 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 0 to100 fluoresce...

Страница 155: ...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 t...

Страница 156: ...o 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 Exporting raw data and analysis data can also be performed...

Страница 157: ...art to Clipboard This copies the graph image to the clipboard Edit Chart in TeeChart Office This opens the graph directly in TeeChart Office for editing see Section 6 4 Print This prints the graph Dig...

Страница 158: ...the left mouse button and dragging the mouse pointer The following options appear Select Only These Samples Samples outside the selected area are deselected Select Only These Samples Samples outside...

Страница 159: ...e work bench area clean and free from dust and sheets of paper The air inlet of the Rotor Gene Q MDx is at the bottom and loose material such as paper or dust may compromise performance To avoid dust...

Страница 160: ...with a lint free cloth dampened with PCR grade water to remove traces of bleach 8 3 Rotor Gene Q repair For repair or service of the Rotor Gene Q contact QIAGEN Technical Services at https www qiagen...

Страница 161: ...on 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 beco...

Страница 162: ...the bottom of the Rotor Gene Q MDx chamber 2 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 MDx and click into...

Страница 163: ...Q MDx CE User Manual 02 2022 163 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...

Страница 164: ...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 adjustment should be required If further...

Страница 165: ...st defines the melting temperature Tm 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 prese...

Страница 166: ...validation Screening for loss of heterozygosity DNA fingerprinting Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Mutation discovery Determining the r...

Страница 167: ...us DNA Polymerase to avoid unspecific amplification products and provide reliable results Note All QIAGEN HRM kits and reagents are indicated for use with Rotor Gene Q instruments only for the applica...

Страница 168: ...r subunit 14B was analyzed on the Rotor Gene Q using 10 ng genomic DNA of different genotypes and the Type it HRM Kit Homozygous wild type AA homozygous mutant GG and heterozygous AG samples are shown...

Страница 169: ...ed DNA For technical details consult the EpiTect HRM PCR Handbook A B Quantitative methylation analysis by HRM Various ratios of methylated and unmethylated DNA APC adenomatosis polyposis coli were an...

Страница 170: ...ke 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...

Страница 171: ...eau 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 sho...

Страница 172: ...commended keeping the amount of template used consistent between samples Spectrophotometric analysis for determining DNA concentration and purity is recommended We recommend QIAGEN kits for sample pre...

Страница 173: ...is used Ensure that the locking ring is in place and the Locking Ring Attached checkbox is checked before proceeding to the next step 3 Set the run details Enter the operator name optional and add any...

Страница 174: ...utton to modify the times and temperatures of the reaction 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 K...

Страница 175: ...Rotor Gene Q MDx CE User Manual 02 2022 175 6 Modify the cycling to suit the amplicon 7 Ensure fluorescence data will be acquired Acquire data to the green channel at the end of the anneal step...

Страница 176: ...tic 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 t...

Страница 177: ...Rotor Gene Q MDx CE User Manual 02 2022 177 10 Start the run by clicking Start Run and save the run file to your computer...

Страница 178: ...responding 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...

Страница 179: ...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 shape chan...

Страница 180: ...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 Region 2 of the melt plo...

Страница 181: ...Graph window displays the normalized melt curves Samples can also be viewed as a difference plot against one of the controls 4 Click on the Genotypes button to define the genotypes Input each genotyp...

Страница 182: ...beled Mutation 1 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 whi...

Страница 183: ...Technical Services solve the problem When contacting QIAGEN Technical Services about errors please have the following information ready Rotor Gene Q MDx serial number type and version Software version...

Страница 184: ...if the reaction was set up correctly Run a positive control experiment i e an assay that is known to provide results Plots look jagged Poor or no amplification Check if the correct protocols and reag...

Страница 185: ...ved from the instrument are not in the correct order Further investigations are required by a QIAGEN Field Service Specialist to diagnose the problem with the instrument Please contact your distributo...

Страница 186: ...being used by another application Close any applications such as communications or synchronization software and then retry This error occurs when the software tries to connect to the machine on the co...

Страница 187: ...ing is currently disabled due to a previous error Archived logs cannot be viewed until the software has been restarted 12 Not all samples could be normalised as the fluorescent level was too low 13 On...

Страница 188: ...efore continuing 42 Your run could not be saved to its default location On the following window select an alternative location to save your run 43 Your settings have been saved Click OK to close the s...

Страница 189: ...5 You have not defined any negative controls You must define negative controls for each channel you are analysing 66 You have not defined any NTC controls You must define NTC controls for each group H...

Страница 190: ...ity 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 Gain Op...

Страница 191: ...ace of operation For indoor use only Pollution level 2 Environmental class 3K2 IEC 60721 3 3 3M2 IEC 60721 3 3 13 2 Transportation conditions Air temperature 25 C to 60 C 13 F to 140 F in manufacturer...

Страница 192: ...C 95 F to 210 2 F 50 C to 99 C for cycling applications Temperature accuracy 0 5 C calibrated using Rotor Disc OTV procedure Temperature resolution 0 02 C smallest programmable increment Temperature u...

Страница 193: ...ursuant to Part 15 of the FCC Rules and meets all requirements of the Canadian Interference Causing Equipment Standard ICES 003 for digital apparatus These limits are designed to provide reasonable pr...

Страница 194: ...d IEC 61326 2 6 QIAGEN GmbH Germany is not responsible for any radio television interference caused by unauthorized modifications of this equipment or the substitution or attachment of connection cabl...

Страница 195: ...ual 02 2022 195 14 3 Declaration of Conformity Name and address of the legal manufacturer QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden Germany An up to date Declaration of Conformity can be requested fro...

Страница 196: ...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 Recycling can be provided by QIAGEN upon re...

Страница 197: ...the duration of the original warranty period and in no case beyond the original expiration date of original warranty unless authorized in writing by an officer of the Company Read out devices interfa...

Страница 198: ...ement 2 2 You may only make copies of the Software only as necessary for backup purposes or when copying is an essential step in the authorised use of the Software You must reproduce all copyright not...

Страница 199: ...you only that a If the software is supplied on CD ROM the CD ROM is free from defects in materials and workmanship under normal use for a period of ninety days from the date of purchase We will replac...

Страница 200: ...AGES OR LOSSES EVEN IF QIAGEN SHALL HAVE BEEN INFORMED OF THE POSSIBILITY OF SUCH DAMAGES IN ANY CASE QIAGEN S ENTIRE LIABILITY UNDER THIS AGREEMENT WILL BE LIMITED TO THE LICENCE FEE PAID BY YOU FOR...

Страница 201: ...tions and CT values of the standards are used to construct a model in the form y Mx B 15 1 1 Confidence intervals for calculated concentrations We use the following confidence interval 100 1 for an es...

Страница 202: ...single standard The extra replicates reduce the uncertainty due to the unknown not forming part of the linear model 15 1 2 Confidence intervals for CT values We assume that error in replicate CT valu...

Страница 203: ...y on parts and labor 9002032 Rotor Gene Q MDx 6plex Real time PCR cycler with 6 channels blue green yellow orange red crimson including laptop computer software accessories 1 year warranty on parts an...

Страница 204: ...s 981604 Rotor Disc 72 Starter Kit Kit includes 3 Rotor Disc 72 packs Rotor Disc Heat Sealer Rotor Disc Heat Sealing Film Rotor Disc 72 Rotor and Locking Ring Rotor Disc 72 Loading Block Rotor Disc Pi...

Страница 205: ...es 0 2 ml requires 36 Well Rotor Locking Ring 9018907 36 Well Rotor Locking Ring For locking PCR Tubes 0 2 ml in the 36 Well Rotor 9018906 Loading Block 96 x 0 2 ml Tubes Aluminum block for manual rea...

Страница 206: ...Rotor Gene Q MDx CE User Manual 02 2022 206 17 Document Revision History Date Changes R1 February 2022 Initial release...

Страница 207: ...p Symantec Symantec Corporation TeeChart Steema Software SL Yakima Yellow Nanogen Inc Registered names trademarks etc used in this document even when not specifically marked as such are not to be cons...

Страница 208: ...Rotor Gene Q MDx CE User Manual 02 2022 208 Ordering www qiagen com contact Technical Support support qiagen com Website www qiagen com HB 3090 001 02 2022...

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