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RIDA

CYCLER

Version 1.0

User manual

12.3.1 Efficiency calculation

The method utilizes the Cycling Analysis feature to determine Cq values and plots them (

-axis)

against the log of the given concentration (x-axis) for each standard annotated in the Samples
editor. A line of best fit is generated for the data plot from which the slope of the line is
determined.

Fig. 55: Efficiency calculation

Efficiency: From the slope of the line the efficiency is calculated using the equation

and is reported in a summary window as a value from 0 to 1 along with the r-squared (R

) value,

Equation of the line, which includes the gradient of the line (

), and

-intercept.

Содержание Rida Cycler

Страница 1: ...R Biopharm AG RIDA CYCLER User Manual BILD...

Страница 2: ...RIDA CYCLER Version 1 0 User manual 2...

Страница 3: ...RIDA CYCLER Version 1 0 User manual 3...

Страница 4: ...express prior written consent Subject to technical changes Technical changes deviations in illustrations and errors reserved 2018 R Biopharm AG Darmstadt User manual RIDA CYCLER ZRCYCLER Version 1 0 2...

Страница 5: ...RIDA CYCLER Version 1 0 User manual 5 User manual...

Страница 6: ...d 19 5 1 Loading tubes 19 5 2 Removing tubes 22 6 Software overview 22 6 1 Tool bar 23 6 1 1 Help icon 23 6 1 2 Open run files 24 6 1 3 Instrument icon 24 6 1 4 Instrument Communication Icon 26 6 2 Fi...

Страница 7: ...a template 52 11 During a run 52 11 1 Modifying a profile during a run optional 53 11 2 Data 53 11 3 Messages 54 12 Analysis 54 12 1 Cycling analysis 55 12 1 1 Graph types 55 12 1 2 Cycling analysis p...

Страница 8: ...Instrument 73 EC Declaration of conformity CE Mark 73 Type Plate Symbols 74 Disposal Information 74 Annex 1 Temperature verification system TVS 75 Annex 2 Error messages and warnings 76 Annex 3 Toshib...

Страница 9: ...accessories or external equipment other than that specified Safety warnings must be adhered to at all times to avoid risk in personal injury and or damage to the instrument If the equipment is used i...

Страница 10: ...RIDA CYCLER Version 1 0 User manual 10 ATTENTION Follow the instructions to ensure optimal instrument performance...

Страница 11: ...rning Do not clean the chamber with flammable liquids The chamber can reach temperatures above 100 C 212 F Any flammable liquids in the chamber could be a fire risk Hot Surface In the event of a user...

Страница 12: ...RIDA CYCLER Version 1 0 User manual 12 Keep the side vents free from obstruction to prevent interference with the cooling of the instrument...

Страница 13: ...s inside the instrument Service should only be performed by an authorized party Intended use The RIDA CYCLER instrument is intended to be used to perform qPCR for molecular biology research applicatio...

Страница 14: ...ptor into the back of the instrument To avoid electrical arcing ensure that the power adapter is not plugged into a wall socket before plugging into the instrument Plug the power cord into a wall sock...

Страница 15: ...RIDA CYCLER Version 1 0 User manual 15 Ensure that the PC has Bluetooth enabled if this is the preferred connection...

Страница 16: ...cted instruments are now ready to be used 1 3 Updating software Please check the website www r biopharm com periodically to see if new software and firmware updates are available or contact your R Bio...

Страница 17: ...d shut during a run to protect the user from moving parts Fig 2 RIDA CYCLER Top view 4 Lid Provides access to the rotor 5 LED indicator When illuminated blue indicates that the instrument is powered O...

Страница 18: ...trument Mic SBS robotic loading block Art No ZRC MIC SBSLB SBS dimension loading block to fit most liquid handling systems Loads 2 x 48 well Mic racks Fig 4 Consumables and accessories 4 LED Indicator...

Страница 19: ...correctly oriented into the loading block and instrument Fig 5 Closing the tubes Use the provided loading rack to pipette reagents and samples into each tube The tubes are pre packed into a loading ra...

Страница 20: ...RIDA CYCLER Version 1 0 User manual 20 Fig 6 Loading rack set up...

Страница 21: ...d water tubes in unused wells No tubes empty tubes and tubes with different volumes of liquid all have different thermal loads on the metal rotor Variations in thermal load around the rotor can cause...

Страница 22: ...the lid for at least 5 minutes until the chamber has cooled The rotor within the chamber could be above 40 C 104 F To avoid personal injury do not touch the rotor for at least 5 min Remove the tube c...

Страница 23: ...the tool bar Instrument Communication Detect available instruments via USB or Bluetooth 6 1 1 Help icon The Help icon is used to access the following options Fig 10 Help icon overview RIDA CYCLER Manu...

Страница 24: ...he type of communication achieved The serial number or name of the instrument is displayed next to the communication symbol The status of the instrument is also displayed beneath the name Idle Instrum...

Страница 25: ...RIDA CYCLER Version 1 0 User manual 25...

Страница 26: ...tware upgrades Temperature Verification Allows you to determine if the instrument is within specification using the Temperature Verification System TVS See Appendix A on how to operate the TVS This op...

Страница 27: ...e associated tab Use the down arrow to view files that might be out of view if multiple tabs are open at the one time Fig 15 File tab overview 6 3 Navigator bar To the left hand side of the main user...

Страница 28: ...1 0 User manual 28 Fig 16 Navigator bar 6 4 File active windows In the central area of the user interface are segmented windows that are active for a specific section of the navigator bar Fig 17 Cycl...

Страница 29: ...RIDA CYCLER Version 1 0 User manual 29...

Страница 30: ...an and standard deviation or as individual wells 6 4 1 CSV Export or Copy to Clip Board Each Result table and Samples editor will have two options for copying the data for easy export to third party s...

Страница 31: ...lication purposes For larger posters e g A0 the images may appear slightly grainy Fig 18 Result image as bitmap 6 4 4 Result Table Organization Each column can be sorted in both Ascending and Descendi...

Страница 32: ...area to view more detail within the graph To zoom back out double click anywhere on the graph Pan Combining the Zoom function with Pan allows you to move the display around a magnified field of view...

Страница 33: ...and displaying the name of the sample in a small text box along with the well number The sample bar in the Samples selector is also highlighted in black Similarly hovering over a sample in the Samples...

Страница 34: ...RIDA CYCLER Version 1 0 User manual 34 View icon located on the sample selector There is also an option to Show All or Hide All...

Страница 35: ...mples Selector Grouping icon to view the sample selector based on Assay Sample Name or None well order Fig 25 Sample grouping This option makes it easier to view or hide samples based on grouped types...

Страница 36: ...emplate for the new run from the drop down list Fig 27 Template window 8 1 Adding assays Select the Assays required for the run by selecting the Add button You can select from any library of assays di...

Страница 37: ...Fig 29 Shortcut library You can create a new assay library Shortcut Select the button next to Personal Shortcuts then browse and select the location of the new directory you wish to use as a personal...

Страница 38: ...le will be adjusted to match the run This may also add melt steps to the run s profile if required by the assay Add Without Modification The selected assay will be added without modification The assay...

Страница 39: ...ual 39 8 3 Updating Changes to an Assay Further changes in the Information fields can be made to the assay These changes can be saved to a new or existing Assay file by clicking on the Save icon locat...

Страница 40: ...ure that the lid is closed prior to starting the run A lid sensor will detect if the lid is open and will prevent the instrument from starting while a warning will notify you of the fact Double check...

Страница 41: ...ersion 1 0 User manual 41 The instrument will automatically lock the lid centrifuge the samples down and then the run profile will begin The LED indicator will turn green to notify a user the instrume...

Страница 42: ...lect from the options below A B C D E F G 2 3 4 5 1 6 H 8 9 10 11 7 12 96 well plate Mic block Left half Right half By column By row Fig 35 Well layout Display wells as either numerical 1 2 3 or alpha...

Страница 43: ...ut different assays will be linked based on the type of analysis chosen You can highlight multiple cells within a column and enter the same characters to annotate replicates Alternatively enter the na...

Страница 44: ...k on the Auto fill icon The names will be sequential based on the first two inputs and the replicates for each will be automatically filled in too Fig 36 sample name options 9 1 5 Type Select the samp...

Страница 45: ...RIDA CYCLER Version 1 0 User manual 45 To change multiple cells at once highlight the cells use the F2 key on your keyboard and then select from the following options...

Страница 46: ...or for contamination of the assay and is helpful in preventing false positives Unlike the NTC the negative control can contain an internal amplification control template to ensure the PCR is working 9...

Страница 47: ...the cells required to complete the dilution series and replicates Click on the Auto fill icon The concentrations will be filled based on the first two inputs and the replicates for each will be automa...

Страница 48: ...cate an assay to a sample will result in the sample not being analyzed Link one Assay to a sample Select the required samples by highlighting the cells in the Assays column Use Ctrl Click to highlight...

Страница 49: ...de a RNA integrity number for each input RNA Comment provide additional information about the samples Fig 40 Select visible sample data columns 9 1 10 Import samples Import sample information from a n...

Страница 50: ...rnings Various warnings will be displayed if annotations have not been completed correctly Some examples include If standards have been selected as Type but no values have been entered into the Standa...

Страница 51: ...RIDA CYCLER Version 1 0 User manual 51 Fig 44 Information window...

Страница 52: ...As button 10 2 Opening a template Install Template Help button on the tool bar In the New button on the tool bar select the template The run will open with all the saved parameters including sample an...

Страница 53: ...run Use this function if you believe you required more cycles to thoroughly complete amplification You can only add another five cycles while the cycling is still running up to a maximum of 60 cycles...

Страница 54: ...uantification Analysis specific parameters are provided for edit and report tables are displayed along with various graphs depending on the analysis type We have taken care to provide the most robust...

Страница 55: ...ny other analysis type that uses cycling data Standard Curve and Absolute Quantification allowing you to adjust parameters associated with generating Cq and efficiency values By selecting Cycling Anal...

Страница 56: ...RIDA CYCLER Version 1 0 User manual 56 Fig 50 Cycling analysis options Linear y axis display the baseline corrected cycling data with the y axis in linear scale by selecting the Linear y axis icon...

Страница 57: ...re first cycles use this setting if there is a significant deviation in the baseline at the start of the run These changes can occur due to many factors including too much template or insufficient den...

Страница 58: ...dyes The default level is set to 5 Auto Exclusion If the sample is excluded its Cq value is not determined and it is not included in the window of linearity calculation Depending on the exclusion set...

Страница 59: ...on samples may also be excluded from the window of linearity calculation if The second derivative maxima occur at the extreme of the data range The logarithm of the normalized fluorescence trace is no...

Страница 60: ...le can be compressed by hiding the individual sample results using the triangle at the top of each replicate row or selecting the Full Collapse option in the Sample table selector for the whole table...

Страница 61: ...esented in the associated parent analysis Fig 54 Floating window Select the delete icon in the top left corner of the floating window to close the floating window 12 3 Standard curve using absolute qu...

Страница 62: ...andard annotated in the Samples editor A line of best fit is generated for the data plot from which the slope of the line is determined Fig 55 Efficiency calculation Efficiency From the slope of the l...

Страница 63: ...le location as a micsc file This file can be imported into any run containing cycling data and used as part of the Absolute Quantification analysis Fig 56 Exporting the standard curve 12 3 2 Standard...

Страница 64: ...ly paired with and will appear beneath the Standard Curve Analysis in the navigator bar Use Cycling Analysis to make modifications to analysis parameters such as Method or Ignore first cycles The same...

Страница 65: ...rve will be displayed on a dashed line Samples outside the limits of a standard curve should be treated with caution To avoid having samples outside the limits of the standard curve ensure that your s...

Страница 66: ...w of the report will be displayed to the right and can be configured to show only certain parts of the run 13 1 Report configuration Each report is divided into two standard sections Run Properties an...

Страница 67: ...rsion of software used The run name will be displayed in the page header Fig 61 Report preview 13 2 1 Run properties The Run Properties section will display the following Name The name of the run File...

Страница 68: ...rations and Assay All 48 samples will be displayed 13 2 3 Analysis Each analysis will be displayed depending on the information provided for the analysis Only the targets selected will be displayed To...

Страница 69: ...page and Last page Export Export the report using one of the available file formats including PDF XLS and Text file Each export will have a set of options to choose from Send Email a report using one...

Страница 70: ...ormation Contains information about the software version file location operator start and finish times along with the run logs Samples The Samples editor is reproduced in Excel format Assays Informati...

Страница 71: ...RIDA CYCLER Version 1 0 User manual 71 Fig 63 Excel workbook...

Страница 72: ...mperature Input Range 35 99 o C min 40 o C when cycling Optical Detectors High sensitivity photodiode per channel Excitation Sources High energy light emitting diode per channel Channels Green Ex 465...

Страница 73: ...ic instrument including the chamber and tube clamp can be decontaminated using a solution of sodium hypochlorite NaOCl A solution containing 1 gL 1 available chlorine will be suitable for sanitation i...

Страница 74: ...for Laboratory Equipment CE Marking The device is in compliance with the essential requirements and other relevant previsions of Low Voltage Directive 2006 95 EC Disposal Information European Union EU...

Страница 75: ...erified to your computer and switch it on Take the TVS out of the protective case and connect it to your computer through USB Open the RIDA CYCLER software Version must be at least 1 0 Verification Pr...

Страница 76: ...and place it back into the protective case Annex 2 Error messages and warnings The following error messages and warnings are possible The selected assay is not compatible with the run The assay s pro...

Страница 77: ...ooth adapter Select the Bluetooth section header and a number of child items will appear In Windows 8 select the Bluetooth VX Module where X is a version number as the Bluetooth adapter In Windows 7 s...

Страница 78: ...will break the signature resulting in a notification by the software The Data Integrity can be found in the Information page of the run file b The ability to generate accurate and complete copies of...

Страница 79: ...rovided by organization document management system see note below h Use of device e g terminal checks to determine as appropriate the validity of the source of data input or operational instruction Th...

Страница 80: ...RIDA CYCLER Version 1 0 User manual 80...

Страница 81: ...alating LC Green 455 495 HRM dye SYTO 9 483 503 HRM dye FAM best 494 515 Conjugated label SYBR Green I 494 521 Intercalating RiboGreen 500 520 RNA label PicoGreen 502 523 ds DNA label Eva Green 503 52...

Страница 82: ...RIDA CYCLER Version 1 0 User manual 82 Quasar 705 690 705 x Conjugated label...

Страница 83: ...AM TET TAMRA and JOE are trademarks and VIC is a registered trademark of Applera Corporation Foster City CA USA Iowa Black is a registered trademark of Integrated DNA Technologies Inc Coralville IA US...

Страница 84: ...using real time quantitative PCR and the 2 Delta Delta C T Method Methods 25 4 402 8 3 Ramakers C Ruijter J M Lekanne Deprez R H and Moorman A F M 2003 Assumption free analysis of quantitative real t...

Страница 85: ...ation cycle CV Coefficient of variation IAC Internal amplification control LoD Limit of detection NTC No template control PCR Polymerase chain reaction qPCR Real time PCR RIN RNA integrity number RT q...

Страница 86: ...ather than another nonspecific target Use of NTC s helps determine analytical specificity Hydrolysis probes are short oligonucleotides with a fluorescence reporter dye at the 5 end and a quencher mole...

Страница 87: ...LER Version 1 0 User manual 87 Bio Molecular Systems Pty Ltd Unit 5 3 Northward Street Upper Coomera QLD 4209 Australia Phone 61 02 9332 1694 E mail support biomolecularsystems com www biomolecularsys...

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