Table of Contents
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QuantStudio
USER GUIDE
for use with:
™
QuantStudio
7 Pro Real-Time PCR System
™
QuantStudio
6 Pro Real-Time PCR System
™
QuantStudio
5 Real-Time PCR System
™
QuantStudio
3 Real-Time PCR System
™
QuantStudio
1 Real-Time PCR System
Publication Number MAN0018202
Revision F.0
For Research Use Only. Not for use in diagnostic procedures.
™
Design and Analysis v2
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Table of Contents
Related Manuals for ThermoFisher Scientific QuantStudio Series
Summary of Contents for ThermoFisher Scientific QuantStudio Series
- Page 1 ™ QuantStudio Design and Analysis v2 USER GUIDE for use with: ™ QuantStudio 7 Pro Real-Time PCR System ™ QuantStudio 6 Pro Real-Time PCR System ™ QuantStudio 5 Real-Time PCR System ™ QuantStudio 3 Real-Time PCR System ™ QuantStudio 1 Real-Time PCR System Publication Number MAN0018202 Revision F.0 For Research Use Only.
- Page 2 Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256 For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC.
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Page 3: Table Of Contents
Contents ■ CHAPTER 1 About the software ........7 Compatible instruments . - Page 4 Contents ■ CHAPTER 4 Review and analyze data ....... . 29 Workflow: General procedures to review the primary analysis results .
- Page 5 Contents Review Replicate Group results ..........48 Configure general plot settings .
- Page 6 Contents ■ APPENDIX A Troubleshooting ......... 62 ■...
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Page 7: Chapter 1 About The Software
About the software ™ QuantStudio Design and Analysis v2 is used to set up instrument runs on and ™ analyze data generated with QuantStudio real‑time PCR systems (see “Compatible instruments” on page 7). Compatible instruments Use the software to create plate files to run on the following instruments: •... - Page 8 Chapter 1 About the software Compatible data files ™ ™ • QuantStudio 12K Flex Real-Time PCR System (except OpenArray format) • StepOnePlus ™ Real-Time PCR System ™ • ViiA 7 Real-Time PCR System • 7500/7500 Fast Real-Time PCR System • 7900HT Real-Time PCR System Note: To convert a legacy data file into the updated file format, open the data file, then click Actions4Save As.
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Page 9: Chapter 2 Workflow
Workflow Set up a plate file (page 10) Select a system template or existing plate file to set up a new plate file (page 10) ▼ Confirm or edit run method (page 12) ▼ Confirm or edit plate setup (page 17) ▼... -
Page 10: Chapter 3 Set Up A Plate File
Set up a plate file Select a system template or existing plate file to set up a new plate file A new plate file must be created from a system template or a previously created plate file. For more information about system templates and plate files, see “About system templates and plate files”... - Page 11 Chapter 3 Set up a plate file Select a system template or existing plate file to set up a new plate file 3. Navigate to, then select a system template or plate file. Description System Contains system templates, non-editable plate files that are Templates included with the software.
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Page 12: Confirm Or Edit Run Method
Chapter 3 Set up a plate file Confirm or edit run method Confirm or edit run method Run method Edit run method elements in the Run Method tab. elements Stage Add/remove stage Step within a stage Add/remove step Temperature ramp rate of a step Number of PCR cycles Temperature of a step PCR stage pause cycle... -
Page 13: Edit Temperature Ramp Rate, Temperature, And Time Length For A Step
Chapter 3 Set up a plate file Confirm or edit run method 3. (Optional) To add the master mix to the reagents table in the Plate Setup tab, select Append selected master mix to reagent table. The master mix is added to the Reagent Table in the Plate Setup tab (see “Edit reagent information”... -
Page 14: Add, Edit, Or Remove A Pause Cycle In A Pcr Stage
Chapter 3 Set up a plate file Confirm or edit run method 3. Select the type of stage from the list. Option Description Hold • Multiple hold stages can be added. Pre-Read • Only one pre-read stage can be added. •... -
Page 15: Edit The Ramp Increment For The Melt Curve Dissociation Step
Chapter 3 Set up a plate file Confirm or edit run method Edit the ramp 1. In the Run Method tab, in the Melt stage, click the name of the ramp increment increment for method, then select one of the following options: the melt curve Option Description... -
Page 16: Enable, Edit, Or Disable Veriflex Zones
Chapter 3 Set up a plate file Confirm or edit run method 7. Click Save. A PCR step with an Auto Delta setting applied to it is denoted with 8. (Optional) To remove Auto Delta, deselect Enable Auto delta. Enable, edit, or ™... -
Page 17: Confirm Or Edit Plate Setup
Chapter 3 Set up a plate file Confirm or edit plate setup Confirm or edit plate setup Select plate • Select plate wells in the (Grid View). wells Action Select a single well Click a well in the plate Select multiple wells Click-drag in the plate Select contiguous wells Shift-click wells in the plate... - Page 18 Chapter 3 Set up a plate file Confirm or edit plate setup Paste samples, targets, or SNP assays in the plate layout ™ You can copy samples, targets, or SNP assays information from an Excel spreadsheet, then paste it into the plate layout grid. 1.
- Page 19 Chapter 3 Set up a plate file Confirm or edit plate setup 2. (Optional) Edit the sample color and sample type (see “Edit sample color and sample type” on page 20). Note: The software automatically assigns a task to the target or SNP assay based on the sample type in a well (see “(Optional) Edit the task assigned to a target or SNP assay in one or more wells”...
- Page 20 Chapter 3 Set up a plate file Confirm or edit plate setup Edit sample color and sample type The software automatically assigns a task to the target or SNP assay based on the sample type in a well (see “(Optional) Edit the task assigned to a target or SNP assay in one or more wells”...
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Page 21: Add Targets Or Snp Assays And Assign To Plate Wells
Chapter 3 Set up a plate file Confirm or edit plate setup 5. (Optional) Edit the custom attribute name or color. a. Select the custom attribute tab. b. In the table, click a field to edit. Add targets or ™ Import plate setup from TaqMan files SNP assays... - Page 22 Chapter 3 Set up a plate file Confirm or edit plate setup Import an Assay Information File (AIF) ™ An Assay Information File (AIF) is provided with every TaqMan assay order. An AIF does not include sample information. Prior to plate setup, download the AIF for your order at thermofisher.com/ taqmanfiles.
- Page 23 Chapter 3 Set up a plate file Confirm or edit plate setup The copied information populates the plate layout, Samples Table, and Targets Table or SNP Assays Table. To complete plate setup, edit the Samples Table, and Targets Table or SNP Assays Table if needed.
- Page 24 Chapter 3 Set up a plate file Confirm or edit plate setup Option Description Target or SNP assay previously defined • Select the target or SNP assay from the dropdown list. • Select the checkbox of the target or SNP assay in the Targets table or SNP Assays table.
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Page 25: Edit Reagent Information
Chapter 3 Set up a plate file Confirm or edit plate setup Task Description Blocked IPC The well contains an IPC blocking agent, which blocks amplification of the IPC. Positive 1/1 The well contains a sample homozygous for allele 1. Positive 2/2 The well contains a sample homozygous for allele 2. -
Page 26: Set Up The Standard Curve
Chapter 3 Set up a plate file Confirm or edit plate setup Set up the A standard curve is only required for standard curve or relative standard curve standard curve analysis. See the appropriate analysis module for more information. Note: ·... -
Page 27: Add Biogroups And Assign Samples
Chapter 3 Set up a plate file Edit plate file or data file information Add biogroups Biogroups, or Biological Replicate Groups, are reactions that contain identical and assign components and volumes, but evaluate separate samples of the same biological samples source. -
Page 28: Review The Plate File And Save
Chapter 3 Set up a plate file Review the plate file and save Review the plate file and save 1. In the Run Summary tab, review the run method selections, then edit if needed (see “Confirm or edit run method” on page 12). 2. -
Page 29: Chapter 4 Review And Analyze Data
Review and analyze data We recommend that you analyze data using the default analysis settings. If the default analysis settings are not suitable for the data, modify the analysis settings, then reanalyze the data. For information about general procedures to analyze data in the Quality Check tab, see Chapter 6, “General procedures to analyze data in the Quality Check tab”. -
Page 30: Open A Data File
Chapter 4 Review and analyze data Open a data file Open a data file To import data files to your Connect account, click (DataConnect) on the left side of the screen. 1. In the home screen, click Open File…. 2. Navigate to, then open a data file. •... -
Page 31: Review The Amplification Status For Each Well
Chapter 4 Review and analyze data Review results in the Amplification Plot Figure 1 Typical amplification plot A typical amplification curve has four distinct sections: Baseline Linear phase Exponential (geometric) phase Plateau phase Review the Note: Amp Status is only applicable for analysis that includes a PCR stage. amplification In the Quality Check tab, in the Well Table, review the amplification status of each status for each... - Page 32 Chapter 4 Review and analyze data Review results in the Amplification Plot 3. Review the threshold values to determine if editing is necessary. A threshold set above or below the optimum can increase the standard deviation of the replicate groups. Table 1 Examples of threshold settings Threshold setting Example...
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Page 33: Review Or Edit Baseline Settings In The Amplification Plot
Chapter 4 Review and analyze data Review results in the Amplification Plot Review or edit 1. In the Quality Check tab, in the plot pane, select Amplification Plot from the baseline dropdown list. settings in the 2. In the plot pane, click (Settings), then make the following selections: Amplification Plot... -
Page 34: Identify And Omit Outliers From Analysis
Chapter 4 Review and analyze data Identify and omit outliers from analysis 4. In the plot pane, click (Settings), then maker the following selections in the Y Axis tab. a. Deselect Auto‑adjust range. b. Enter Minimum value of –1. c. Enter Maximum value of 2. Figure 3 Example amplification plot of negative controls The linear plot displays the amplification plot for negative controls as smooth lines. -
Page 35: Review Results In The Multicomponent Plot
Chapter 4 Review and analyze data Review results in the Multicomponent Plot Review results in the Multicomponent Plot If no data are displayed in the Quality Check tab, or if reanalysis is required, click Analyze. 1. In the Quality Check tab, in the plot pane, select Multicomponent Plot from the dropdown list. -
Page 36: Review Results In The Raw Data Plot
Chapter 4 Review and analyze data Review results in the Raw Data Plot Review results in the Raw Data Plot For more information about the Raw Data plot, see “Raw Data Plot overview” on page 60. If no data are displayed in the Quality Control tab, or if reanalysis is required, click Analyze. -
Page 37: Review Qc Alerts In The Well Table
Chapter 4 Review and analyze data Review QC alerts in the Well Table Figure 6 Example Melt Curve Plot Review QC alerts in the Well Table If no data are displayed in the Results tab, or if reanalysis is required, click Analyze. 1. -
Page 38: View Or Edit Qc Alerts Settings
Chapter 4 Review and analyze data Edit primary analysis settings 1. Open a plate file or data file, then click Actions4Primary Analysis Setting..2. In the General tab, select an option from the PCR Stage/Step dropdown list. 3. Select an option from the Algorithm Settings dropdown list. 4. -
Page 39: View Or Edit Melt Analysis Settings
Chapter 4 Review and analyze data Edit primary analysis settings c. Select the Sample Type from the dropdown list, then select the Target from the dropdown list. d. In the right panel, select the acceptance criteria for each sample type and target combination. -
Page 40: View Instrument Calibration Results
Chapter 4 Review and analyze data View instrument calibration results 1. Open a data file, then click Actions4Use Settings from Another File..2. Navigate to the plate file or data file that contains the desired analysis settings. 3. Select the file, then click Open. The data is reanalyzed using the new analysis settings. -
Page 41: Review Dye Calibration Results
Chapter 4 Review and analyze data Override the calibration data Review Dye 1. Review the calibration properties, including calibration status, in the menu bar. calibration 2. Select a Dye row in the Calibration table to view the corresponding analysis results data plot. -
Page 42: Perform Additional Analysis
Chapter 4 Review and analyze data Perform additional analysis Perform additional analysis Perform additional analysis using one of the following options: Option Description ™ To perform additional analysis using QuantStudio Design and Analysis v2, select an analysis module (see “Select an ™... -
Page 43: Chapter 5 Export Results
Export results For information about export settings, see Chapter 9, “Manage export settings”. Export the Well Table 1. In the Quality Check tab, in the Well Table, click (Actions)4Export. 2. Name the file, navigate to the desired folder location, then click Save. ™... -
Page 44: Export Data In The Quantstudio ™ File Format
Chapter 5 Export results Export data in the QuantStudio ™ file format ™ Export data in the QuantStudio file format Export analyzed data for further analysis. Save the analyzed data file before exporting the results (see “Save a plate file or data file”... - Page 45 Chapter 5 Export results Generate a report 5. In the Report Content pane, select the content to include in the report: Report Content Description Displays a summary of the experiment, including the following information: • Analysis Module • Bar Code •...
- Page 46 Chapter 5 Export results Generate a report Report Content Description Amplification Plot (Rn) Displays the Amplification Plot (Rn vs Cycle) Amplification Plot (dRn) Displays the Amplification Plot (dRn vs Cycle) Displays the Melt Curve Plot (Derivative Reporter vs Temperature) This option only Melt Curve Plot displays if the Run Method includes a Melt Curve stage.
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Page 47: Chapter 6 General Procedures To Analyze Data In The Quality Check Tab
General procedures to analyze data in the Quality Check tab For detailed procedures, see Chapter 4, “Review and analyze data”. View the post-run summary 1. Open the data file. 2. In the Run Summary tab, view a summary of the run, including the following information: •... -
Page 48: Review Individual Well Results In The Well Table
Chapter 6 General procedures to analyze data in the Quality Check tab Review individual well results in the Well Table Review individual well results in the Well Table In the Quality Control tab, view results for individual wells in the Well Table. •... -
Page 49: Chapter 7 Manage Plate Files And Data Files
Manage plate files and data files About system templates and plate files A plate file contains the information that is necessary to perform an instrument run. A system template is a non-editable plate file that is included with the software. Opening a system template automatically generates a new plate file that can be edited, then saved (see “Select a system template or existing plate file to set up a new plate file”... -
Page 50: About Data Files
Chapter 7 Manage plate files and data files About data files About data files A data file contains the information from the plate file that was used to perform the instrument run. A data file can also contain the following information: Information type Properties •... -
Page 51: Add A Plate File To My Plate Files
Chapter 7 Manage plate files and data files Add a plate file to My Plate Files Note: A blue checkbox ( ) indicates that the function cannot be edited. 3. Enter and confirm a password, then click Restrict. Note: Record the password because lost passwords cannot be recovered. A lock icon ( ) appears next to the file name in the software menu bar to indicate that restricted editing is enabled. -
Page 52: Chapter 8 Manage Analysis Modules
Manage analysis modules To see all of the analysis modules that are installed, click Settings4Plugins. About analysis modules Analysis modules are plugins that enable additional data analysis using ™ QuantStudio Design and Analysis Software v2. • To see all of the analysis modules that are installed, click Settings4Plugins. -
Page 53: Select An Analysis Module
Chapter 8 Manage analysis modules Select an analysis module Select an analysis module The analysis module selection can be selected in either the plate file or the data file. 1. In a plate file or data file, click Actions4Analysis Modules. 2. -
Page 54: Chapter 9 Manage Export Settings
Manage export settings About export settings Export settings designate the data to include in the exported results. The software includes several non-editable export settings files in the Export Settings library (click System4Export Settings to view. To edit the default settings, you must create a new export settings file (see “View or edit export settings”... -
Page 55: View Files In The Export Settings Library
Chapter 9 Manage export settings View files in the Export Settings library 6. To edit the export settings for analysis module data, select the analysis module from the Analysis Module dropdown list. 7. Edit the analysis module data export settings (see step 5). 8. -
Page 56: Import An Export Settings File
Chapter 9 Manage export settings Import an export settings file Import an export settings file Import a previously downloaded export settings file (see “Download an export settings file” on page 55). 1. In any screen, click System4Export Settings. 2. Click Actions4Import Setting. 3. -
Page 57: Chapter 10 About Data Analysis
About data analysis About the quantification cycle (C The quantification cycle (C ) is used for gene expression metrics quantification analysis. Algorithm-specific calculations of C values are used as the primary input values for quantification analysis. Algorithm Description is calculated using the PCR cycle number at which the fluorescence signal meets Baseline Threshold the threshold in the amplification plot. -
Page 58: Primary Analysis Settings Overview
Chapter 10 About data analysis Primary analysis settings overview Primary analysis settings overview settings The default C settings are appropriate for most applications. Edit the settings for overview analysis of not typical or unexpected run data. Note: The run data must include a PCR stage to perform C analysis. -
Page 59: Guidelines For Manual Threshold And Baseline Settings For C Analysis
Chapter 10 About data analysis Plots overview Guidelines for Setting Recommendation manual threshold and Threshold Enter a value for the threshold so that the threshold is: baseline • Above the background. settings for C • Below the plateau and linear phases of the amplification curve. analysis •... -
Page 60: Raw Data Plot Overview
Chapter 10 About data analysis Plots overview Three plots are available. Some plots can be viewed as a linear or log graph. Table 3 Amplification Plot types Plot type Description Use to • Identify and examine DRn vs Cycle DRn is the magnitude of normalized irregular amplification. -
Page 61: Melt Curve Plot Overview
Chapter 10 About data analysis Plots overview Melt Curve Plot The Melt Curve Plot displays the melt curve of the amplification products in the overview selected wells. Review the Melt Curve Plot to confirm that the amplification products in a well display a single melting temperature (T ). -
Page 62: Appendix A Troubleshooting
Troubleshooting Observation Possible cause Recommended action High fluorescence signal The reaction volume is not Ensure that reaction volumes in the plate are correct. correct and match the volume that is entered in the Run Method tab. Signals that exceed the limit of Examine the bottom of the reaction plate. -
Page 63: Documentation And Support
Documentation and support Related documentation Document Publication number ™ QuantStudio 6 Pro and 7 Pro Real-Time PCR Systems User MAN0018045 Guide ™ QuantStudio 3 and 5 Real-Time PCR Systems Installation, MAN0010407 Use, and Maintenance Guide ™ QuantStudio 1 Real-Time PCR System Installation, Use, MAN0017853 and Maintenance Guide Customer and technical support... -
Page 64: Index
Index custom attribute, sample 20 add, biogroup 27 additional analysis 42 algorithm settings data file Baseline Threshold Algorithm 58 information 49, 50 Relative Threshold Algorithm 58 open 30 Amp Status 31 restrict editing 50 Amplification Plot save 50 baseline 30 search 51 baseline setting 33 tag 51... - Page 65 Index ramp increment 15 related documentation 63 settings 59 Relative Threshold Algorithm 58 Melt Curve Plot report, generate 44 overview 61 restrict editing, plate file or data file 50 view and assess 36 results, export 43 melt settings, configure 39 review method Amplification Plot 30...
- Page 66 thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 15 April 2020...
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