QuantStudio Design And Analysis Software - Thermo Fisher Scientific

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QuantStudio Design and Analysis SoftwareUSER GUIDEGetting started with design and analysis of experiments in thedesktop software v1.2.xfor use with: QuantStudio 3 and 5 Real-Time PCR SystemsPublication Number MAN0010408Revision B.0For Research Use Only. Not for use in diagnostic procedures.

The information in this guide is subject to change without notice.DISCLAIMERTO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.NOTICE TO PURCHASER: DISCLAIMER OF LICENSEPurchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagentor kit rights under patent claims owned or otherwise controlled by Life Technologies Corporation, either expressly, or by estoppel.Corporate entityLife Technologies Carlsbad, CA 92008 USA Toll Free in USA 1.800.955.6288TrademarksAll trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of RocheMolecular Systems, Inc., used under permission and license. 2015 Thermo Fisher Scientific Inc. All rights reserved.

ContentsAbout this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Scope of the guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 CHAPTER 1 General procedures to set up and run experiments . . . . 9Set up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Create or open an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Workflow bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Enter experiment properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Confirm or edit the experiment method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Define samples and targets or SNP assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Assign samples, targets or SNP assays, and tasks to wells . . . . . . . . . . . . . . . . . . . . . . 13Save the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Shortcuts for experiment setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Guidelines for handling samples and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Guidelines for setting up the reactions in the plates or tubes . . . . . . . . . . . . . . . . . . . .17171717Run an experiment from the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 CHAPTER 2 General procedures to review results . . . . . . . . . . . . . . . . . . 21Overview of the Results tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22General workflow for analysis of experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Guidelines for viewing and analyzing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Assess results in the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Assess the overall shape of the Amplification Plot curves . . . . . . . . . . . . . . . . . . . . . . .Confirm or correct threshold settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Confirm or correct baseline settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Omit outliers from analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optimize display of negative controls in the Amplification Plot . . . . . . . . . . . . . . . . . . .25252626282929Assess results in the Well Table view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30About the Well Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30QuantStudio Design and Analysis desktop Software User Guide3

ContentsConfirm accurate dye signal using the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . 31About the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31View and assess the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Determine signal accuracy using the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33About the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33View and assess the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Review the flags in the QC Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Configure analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and configure the analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the CT settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the flag settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About Advanced Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .343434353636About overriding the calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Export experiments or results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Options for publishing the analyzed data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Export configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 CHAPTER 3 Getting started with Standard Curve experiments . . . . 38About Standard Curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38383939Set up a Standard Curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General workflow for analysis of Standard Curve experiments . . . . . . . . . . . . . . . . . .About the Standard Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and assess the Standard Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the Standard Curve settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4242424344CHAPTER 4 Getting started with Relative Standard Curve andComparative CT experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45About Relative Standard Curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45454646About Comparative CT experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47474747Relative quantitation: Relative Standard Curve vs Comparative CT . . . . . . . . . . . . . . . . . . . . 48Set up a Relative Standard Curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . 484QuantStudio Design and Analysis desktop Software User Guide

ContentsSet up a Comparative CT experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General workflows for analysis of Relative Standard Curve and ComparativeCT experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and assess the Standard Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the Gene Expression Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About the QC Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About relative quantification settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525253555657CHAPTER 5 Getting started with Genotyping experiments . . . . . . . . . 58About Genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58585959Set up a Genotyping experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General workflow for analysis of Genotyping experiments . . . . . . . . . . . . . . . . . . . . . .About the Allelic Discrimination Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and assess the Allelic Discrimination Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Perform manual calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About call settings for Genotyping experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616162626364CHAPTER 6 Getting started withPresence/Absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66About Presence/Absence experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Compatible PCR options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66666767Set up the Presence/Absence experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Set up and run the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General workflow for analysis of Presence/Absence experiments . . . . . . . . . . . . . . . .About the Presence/Absence Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and assess the Presence/Absence Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About call settings for Presence/Absence experiments . . . . . . . . . . . . . . . . . . . . . . . . .QuantStudio Design and Analysis desktop Software User Guide69697070725

Contents CHAPTER 7 Getting started with Melt Curve experiments . . . . . . . . . . 73About Melt Curve experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Reaction types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Set up a Melt Curve experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Set up Melt Curve reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General workflow for analysis of Melt Curve experiments . . . . . . . . . . . . . . . . . . . . . . .About the Melt Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .View and assess the Melt Curve Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About Melt Curve settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7575757677APPENDIX A Alternative procedures and shortcuts forsetting up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Set up a custom experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79About custom experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79Set up a custom experiment in the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Adjust method parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Method elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Set up specialized temperature zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Edit the ramp increment for the Melt Curve dissociation step . . . . . . . . . . . . . . . . . . . . . . . 82Assign the standard dilutions manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Shortcuts for experiment setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Sample, target, and SNP assay libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Apply a filter to search a library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import to the Sample Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import from the Sample Library to the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import to the Target Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import from the Target Library to the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import to the SNP Assay Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import from the SNP Assay Library to the experiment . . . . . . . . . . . . . . . . . . . . . . . . . .848484848585858686Assign samples using a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About sample definition files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Create a sample definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Import sample information from a sample definition file . . . . . . . . . . . . . . . . . . . . . . . .86868687Assign samples and targets using plate setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87About plate setup files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Import plate setup data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876QuantStudio Design and Analysis desktop Software User Guide

ContentsAssign targets, samples, and biological replicate groups from an *.xls file . . . . . . . . . . . . 88Set up an experiment using templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .About experiment templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Options for working with unlocked templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Options for working with locked templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89899090APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Power on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Transfer files from the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 APPENDIX C Parts and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Kits, consumables, and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Compatible reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9496-Well (0.2-mL) consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95384-well consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9596-Well Fast (0.1-mL) consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 QuantStudio 3 and 5 Systems accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97General-use materials and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98Obtain information from the Help system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102QuantStudio Design and Analysis desktop Software User Guide7

About this guideRevision historyRevisionDateB.0December 2015DescriptionUpdates include: Combination of define and assign functions into asingle Plate tab Display of VeriFlex Zones on plate layout Real-time data monitoring in the Run tab Security, Audit, and E-Signature (SAE) features Implementation of locked workflow Selection of an instrument before starting a run Ability to select multiple targets in the results view Various minor changes to the user interfaceA.0April 2015New document.Scope of the guideThis guide is intended to help users get started with design and analysis ofexperiments in the QuantStudio Design and Analysis desktop Software.The software includes multiple options for many steps. This guide presents a single,straightforward procedure for setup and analysis of each experiment type. Alternativeoptions and shortcuts are included in Appendix A, “Alternative procedures andshortcuts for setting up an experiment“.Also, the guide directs the user to the desktop software Help system for step-by-stepinstructions for procedures that are not routinely performed by the user.8QuantStudio Design and Analysis desktop Software User Guide

1General procedures to set up andrun experiments Set up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Prepare reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Run an experiment from the desktop software . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Set up an experimentThis section describes the general procedures to set up an experiment in the desktopsoftware. For setup information for a specific experiment type, see: “Set up a Standard Curve experiment in the software“ on page 40 “Set up a Relative Standard Curve experiment in the software“ on page 48 “Set up a Comparative CT experiment in the software“ on page 50 “Set up a Genotyping experiment in the software“ on page 59 “Set up the Presence/Absence experiment in the software“ on page 68 “Set up a Melt Curve experiment in the software“ on page 74 “Set up a custom experiment in the software“ on page 80Create or open anexperimentIn the Home screen: Create a new experiment.ToActionCreate anClick Create New Experiment.experiment withoutpreexisting settingsCreate anexperiment from asystem template1. Select Create New Experiment4Template.2. Navigate to and select the desired file, then click Open.Template files are installed with the software in: drive :\Program Files (x86)\AppliedBioSystems\QuantStudio Design & AnalysisSoftware\templates,where drive is the drive on which the software isinstalled. Open an existing experiment.a. Click Open.QuantStudio Design and Analysis desktop Software User Guide9

1Chapter 1 General procedures to set up and run experimentsSet up an experimentb. Navigate to and select the desired file, then click Open.Continue to “Enter experiment properties“ on page 10.Workflow barThe workflow bar is used for navigation through experiment design and analysis.Enter experimentpropertiesIn the Properties tab:1. (Optional) Modify the default experiment Name.The experiment Name determines the file name of the experiment. However,when you save your experiment, changes to the experiment name from theProperties tab do not update the file name. To change the file name after an initialSave4Save as.save, select2. (Optional) Enter or scan a Barcode.Note: Click in the Barcode field before using a barcode scanner.3. (Optional) Enter a User name and Comments.4. Select an Instrument type, Block type, Experiment type, Chemistry (reagents),and Run Mode (Fast or Standard) from the drop-down lists.Note: Selecting the experiment type defines the available options for theexperimental setup. See the “Experiment definitions“ on page 11 for theparameters defined in each experiment type.5. (Optional) Click Manage chemistry details (see “Enter reagent information“ onpage 10).Save or select6. (Optional) Click(.edt) file before continuing.Save4Save as. to save the experimentClick Next to advance to “Adjust method parameters“ on page 81.Enter reagent informationRefer to the software Help system for step-by-step instructions for entering reagentinformation.Confirm or editthe experimentmethodIn the Method tab: (Optional) Adjust the reaction volume. (Optional) Edit the default run method (thermal protocol) (see “Adjust methodparameters“ on page 81).The default run method has been optimized for TaqMan assays and a broadrange of other reagents.10QuantStudio Design and Analysis desktop Software User Guide

Chapter 1 General procedures to set up and run experimentsSet up an experimentDefine samplesand targets orSNP assays1These are general instructions. For specific instructions for each experiment type, seethe appropriate chapter in this guide.Refer to the software Help system for step-by-step instructions for defining samplesand targets or SNP assays.(Optional) SelectAction4Save to Library to save a sample, target, or SNP assay toits respective library.Experiment definitionsThe parameters you can define vary by experiment type.Experiment TypeTargetsSNP enceReference andEndogenousControlsStandard CurveRelative StandardCurveComparative CTMelt CurveGenotypingPresence /AbsenceCustomManually define a sampleIn the Plate tab:1. Click Advanced Setup.2. In the Samples table, clickAdd.3. (Optional) Click a cell to edit Color or Sample Name.in the table header to add a Custom Attribute column.4. (Optional) Clicka. Click the Custom Attribute column header, then edit the header with a newsample attribute.b. Click a Custom Attribute cell in the table, then enter a sample definition.5. (Optional) Clickto delete a sample from the table.QuantStudio Design and Analysis desktop Software User Guide11

1Chapter 1 General procedures to set up and run experimentsSet up an experimentManually define a targetIn the Plate tab:1. Click Advanced Setup.2. In the Targets table, clickAdd.3. (Optional) Click a cell to edit Color, Name, Reporter dye, Quencher, or to addComments.4. (Optional) Clickto delete a target from the table.Manually define a SNP assayOnly Genotyping experiments contain SNP assay options.In the Plate tab:1. Click Advanced Setup.2. In the SNPs table, clickAdd.3. In the New SNP Assay dialog box, enter: SNP Assay Name Color Assay ID Gene Symbol Gene Name NCBI SNP Reference Allele 1 Name or Bases(s), Color, Reporter, and Quencher Allele 2 Name or Bases(s), Color, Reporter, and Quencher Context Sequence Comments4. Click OK.5. (Optional) Clickto delete a SNP assay from the table.Define biological replicate groupsBiological Replicate Groups can be defined in Standard Curve, Relative StandardCurve, Comparative CT, and custom experiments.In the Plate tab:1. Click Advanced Setup.2. In the Biological Replicate Groups table, clickAdd.3. (Optional) Click a cell to edit color, name, or comments.4. (Optional) Click12to delete a biological replicate group from the table.QuantStudio Design and Analysis desktop Software User Guide

Chapter 1 General procedures to set up and run experimentsSet up an experimentAssign samples,targets or SNPassays, and tasksto wells1These are general instructions. For specific instructions for each experiment type, seethe appropriate chapter in this guide.Refer to the software Help system for step-by-step instructions for defining samplesand targets or SNP assays.Note: Only Genotyping experiments contain SNP assay options.Note: You can start a run without well assignments, but the amplification plots aredisplayed only after wells are assigned in the software.Select plate wellsClick the appropriate icon to access the In thePlate La

Run an experiment from the desktop software . 19 Set up an experiment This section describes the general procedures to set up an experiment in the desktop software. For setup information for a specific experiment type, see: "Set up a Standard Curve experiment in the software" on page 40

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