Agilent Aria Real-Time PCR User Manual

Aria Real-Time PCR Software

Always make sure you are using the most recent version of the Aria software. Check the Aria software download website at www.agilent.com/en/ariamx-software-download.

User Manual

Revision H0, March 2021

AriaMx: For Research Use Only. Not for use in diagnostic procedures.

AriaDx: For In Vitro Diagnostic Use

Agilent Technologies

1 Getting Started with the Aria Software 14

The Aria software 15

Getting Started with the Aria Software 16

Introduction to the Aria software 16

Overview of the user interface 18

Home screen 18 Menu toolbar 18 Tabs 18 Left and right panels 18 Home/Notifications/Help icons 19 Getting Started screen 19 Experiment Notes / Project Notes 19

Help access for the Aria software 20

Help system 20 Sample experiments 21

2 Specifying Hardware and Software Settings 22

Update instrument optics 23

Change the default crosstalk correction settings 25

Set software preferences 28

Set default file name configuration 28 Create and apply analysis templates 29

3 Performing Hardware and Software Tests and HRM Calibrations 32

Run an instrument qualification test 33

Run the test 33 About the Qualification Test graphical data 34

Perform an Installation Qualification test 36

Run an HRM calibration plate (HCP) 38

Prepare the plate 38

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Run an HCP 39 If the HCP fails 40

4 Creating/Opening an Experiment 42

Overview of the Getting Started screen 43

Tools available on the Getting Started screen 44 About the Aria file types 45

Quick Start Protocol 46

How to create, set up, run, analyze, and generate reports for an experiment 46

Create a new experiment 49

Create an experiment based on experiment type 49

Create an experiment from a template 50

Create an experiment from a LIMS data file 51

Open an existing experiment 53

Save a copy of an existing experiment 54

Create a template from an existing experiment 55

Convert an experiment to a new experiment type 56

5 Selecting an Experiment Type 58

Overview of Experiment Types 59

Quantitative PCR 59 Comparative Quantitation 59 Allele Discrimination - Fluorescence Probes 60 Allele Discrimination - DNA Binding Dye with High-Resolution Melt 60 User Defined 60

The Quantitative PCR Experiment Type 61

Multiplexing quantitative PCR experiments 61 Well types for Quantitative PCR experiments 62

The Comparative Quantitation Experiment Type 63

Normalizing chance variations in target levels 63

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Determining amplification efficiencies for the targets of interest and normalizer targets 64 Including biological replicates in comparative quantitation 65 Well types for Comparative Quantitation experiments 66

The Allele Discrimination - DNA Binding Dye Experiment Type 67

About HRM calibration plates 67 Well types for Allele Discrimination - DNA Binding Dye experiments 68

The Allele Discrimination - Fluorescence Probe Experiment Type 69

Well types for Allele Discrimination - Fluorescence Probe experiments 70

The User Defined Experiment Type 71

6 Setting Up the Plate 72

Overview of the Plate Setup screen 73

The plate map 74 Well type 74 Well name / Sample name 74 Target information 75 Replicate number 75 Reference dye 75 The Properties panel 76 Additional tools for setting up a plate 76

Import a plate setup 78

Select and view wells in the plate map 79

Select wells in the plate map 79 Unselect wells in the plate map 80 View details of a well or wells 80

Export the plate map image 85

Assign plate properties for a Quantitative PCR DNA Binding Dye experiment 86

Assign well types 86 Assign well names 86 Assign dyes/targets 88 Select a reference dye 89

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Assign replicates 90 Assign quantities to Standard wells 92

Assign plate properties for a Quantitative PCR Fluorescence Probe experiment 94

Assign well types 94 Assign well names 94 Assign dyes/targets 96 Select a reference dye 97 Assign replicates 98 Assign quantities to Standard wells 100

Assign plate properties for a Comparative Quantitation experiment 102

Assign well types 102 Assign well names 103 Assign sample names and biological replicates 104 Assign dyes/targets 107 Select a reference dye 108 Designate the normalizer 108 Assign replicates 109 Assign quantities to Standard wells 111

Assign plate properties for an Allele Discrimination DNA Binding Dye experiment 113

Assign well types 113 Assign well names 113 Assign sample names 115 Assign dyes/targets 117 Select a reference dye 118 Assign replicates 119

Assign plate properties for an Allele Discrimination Fluorescence Probe experiment 122

Assign well types 122 Assign well names 122 Assign sample names 124 Assign dyes/targets 126 Select a reference dye 127 Assign Alleles 128

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Assign replicates 128

Assign plate properties for a User Defined experiment 131

Assign well types 131 Assign well names 131 Assign sample names and biological replicates 133 Assign dyes/targets 135 Select a reference dye 137 Designate the normalizer 137 Assign Alleles 138 Assign replicates 138 Assign quantities to Standard wells 141

7 Setting Up the Thermal Profile 142

Set up the thermal profile 143

Elements of a Thermal Profile 144 Import a thermal profile 146 Edit the thermal profile 147

Export the thermal profile image 159

8 Running and Monitoring Experiments 160

Overview of the Instrument Explorer dialog box 161

Add instruments to your network 162

Add a new instrument based on its IP address 162 Add a new instrument based on its port number 163 View information about an instrument 163

Start, stop, or pause a run 165

Start a run 165 Cancel a run 166 Pause a run 167

Monitor a run 168

Connect to the running instrument 168

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Monitor a run by viewing its progress through the thermal profile 169 Monitor a run by viewing the raw data plots 169 Change the display options for the raw data plots 170 Stop monitoring a run 172

Retrieve run data from the instrument 173

Retrieve data through the network 173 Retrieve data using a USB drive 174

Export instrument data to a CSV file 175

Export instrument data by column 175 Export instrument data by target 175 Export instrument data by wells 175

9 Setting Analysis Criteria 176

Overview of the Analysis Criteria screen 177

Toggle display of plate map wells 178

Select the wells and well types to include in analysis 179

Select wells for analysis using the plate map 179 Select wells for analysis based on well type 179

Select the targets to include in analysis 180

Select which data collection points to analyze 181

Choose a treatment for replicate wells 182

Assign an HRM calibration plate 183

Assign an HCP for new experiments 183 Assign an HCP using the HCP icon 184

10 Viewing Graphical Displays of the Results 186

Overview of the Graphical Displays screen 187

Graphs 187 Result table 188 Display options 188

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Zooming 190

Configure and apply analysis templates 192

Configure an analysis template in the Analysis Term Settings dialog box 193

View the Amplification Plots 197

View data for a single data point 197 Select a fluorescence data type 198 Specify the use of smoothing 198 Adjust the graph properties 199 Adjust the baseline correction settings 199 Adjust the crosstalk correction settings 201 Select the scale (linear or log) of the Y-axis 206 Manually adjust threshold fluorescence values 207 Adjust threshold fluorescence values by altering the algorithm settings 209 Lock or unlock the threshold fluorescence values 210

View the Melt Curve - Raw/Derivative Curve 212

About raw/derivative curves 212 View data for a single data point 213 Select a fluorescence data type 213 Adjust the graph properties 214 Specify the use of smoothing 214 Normalize the fluorescence values 214 Adjust the range of the X-axis 215 Adjust product melting temperature settings 216

View the Melt Curve - Difference Plots 218

About difference plots 218 Select a fluorescence data type 220 Assign the control target 220 View data for a single data point 221 Manually assign Unknowns to a genotype call 221 Adjust the graph properties 224

View the Standard Curve 225

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View data for a single data point 225 Select a fluorescence data type 226 View the R-squared values, slopes, and amplification efficiencies 227 Adjust the graph properties 228 Manually adjust threshold fluorescence values 228 Display and adjust confidence intervals 229

View the Relative Quantity 230

About relative quantities 230 Select a fluorescence data type 231 Set the Y-axis scale for the Relative Quantity chart 231 Add error bars to the Relative Quantity chart 232 Adjust the graph properties 232 Select the algorithm method 232 Enter the amplification efficiencies for the targets 233

View the Allele Determination graph 235

View data for a single data point 235 Select data type and fluorescence type to display 236 Display genotype groups on the graph 237 Adjust the graph properties 238 Adjust the last cycle 238 Rename the genotype groups 239

Customize graph properties 240

Customize graph properties using the short-cut menu 240 Customize graph properties using the Graph Properties dialog box 243

11 Generating Reports and Exporting Results 252

Generate report of results 253

View a preview of the report 253 Select report type 253 Generate the report 253 Configure the report 254 Create or edit report configuration definitions 257

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Export data/results to an Excel, text, LIMS data, or RDML file 260

Configure the file and export data 260 Load a saved data export definition 264 Create or edit data export definitions 265

12 Creating and Setting Up an MEA Project 268

Quick Start Protocol 269

How to create, set up, analyze, and generate reports for a multiple experiment analysis

project 269

Overview of multiple experiment analysis 270

Applications 270 Restrictions 271

Guidelines for Comparing Cq Values Across Experiments 272

Reducing plate-to-plate variability 272 Selecting a method for setting threshold fluorescence levels 273

Create an MEA project 275

Open an existing MEA project 276

Select experiments for a project 277

Add or remove experiments from the project 277 Include or exclude experiments in the project analysis 278

Edit the plate setup of experiments in a project 279

Select an experiment to edit 279 Differentiate between targets across experiments 279 Edit plate properties 280

View the thermal profiles of experiments in a project 281

13 Analyzing Multiple Experiment Analysis Project Results 282

Set analysis criteria for a project 283

Toggle display between one experiment and all experiments 283 Select the wells and well types to include in analysis 283 Select the targets to include in analysis 284

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Select which data collection points to analyze 284 Choose a treatment for replicate wells 285

Overview of the Graphical Displays screen for a project 286

Graphs 286 Result table 287 Display options 287 Zooming 289

Compare amplification plots in a project 291

Compare amplification plots by experiment 291 Compare amplification plots by target 291 Consolidate the amplification plots 292

Compare raw or derivative melt curves in a project 293

Compare raw or derivative melt curves by experiment 293 Compare raw or derivative melt curves by target 294 Consolidate the raw or derivative melt curves 294

Compare standard curves in a project 295

Compare standard curves by experiment 295 Compare standard curves by target 296 Consolidate the standard curves 296

Compare Relative Quantity charts in a project 297

Compare relative quantities by experiment 297 Compare relative quantities by target 298 Consolidate the relative quantities 298

Compare Allele Determination graphs in a project 299

14 Generating Multiple Experiment Analysis Reports and Exporting Results 300

Generate report of MEA project results 301

View a preview of the report 301 Select report type 301 Generate the report 301 Configure the report 302

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Create or edit report configuration definitions 305

Export MEA data/results to an Excel, text, or LIMS data file 308

Configure the file and export data 308 Load a saved data export definition 311 Create or edit data export definitions 312

15 “How-To” Examples for Multiple Experiment Analysis Projects 314

Example 1 315

How to find the initial template quantity of a target in an unknown sample using a standard curve

from a separate experiment 315

Example 2 317

How to normalize target quantities in Unknown and Calibrator wells using a normalizer target from a

separate experiment 317

16 Help for the Aria ET (Electronic Tracking) Software 320

Overview of the Aria ET software 321

Open an experiment in the Aria ET software 323

Open an experiment 323

Import and export experiments in the Aria ET software 326

Import experiments into the database 326 Export experiments from the database 326

Lock or log out of the Aria ET software 328

Lock the program 328 Log out of the program 328

Change your password in the Aria ET software 330

Create a multiple experiment analysis project in the Aria ET software 331

Manage users in the Aria ET software 332

Set account properties for all users 332 Manage user accounts 335

Archive and restore experiments in the Aria ET software 339

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Archive experiments 339 Restore experiments 341

View audit trails and system logs in the Aria ET software 343

View the audit trail logs 343 View the system logs 346

Add and remove databases in the Aria ET software 348

Add an Aria ET database 349 Remove an Aria ET database 350

View transaction logs in the Aria ET software 352

View transaction logs for primary database 352 View transaction logs for an archive database 353

17 Reference Help and Troubleshooting & Support 354

QPCR Glossary 355

Experiment Type and QPCR Detection Chemistry Terms 355 Well-Types 357 Analysis Terms 358

LIMS File Format for Aria Software 361

Plate Setup 361 Experiment Setup 363 Thermal Profile 363 Default Optical Module 364 Threshold Fluorescence 364

Trademarks 366

Troubleshooting and Support 367

Troubleshooting Guide Contact Agilent Technical Support 370

13 Agilent Aria Real-Time PCR Program

1 Getting Started with the Aria Software

The Aria software 15 Getting Started with the Aria Software 16

Introduction to the Aria software 16

Overview of the user interface 18

Home screen 18 Menu toolbar 18 Tabs 18 Left and right panels 18 Home/Notifications/Help icons 19 Getting Started screen 19 Experiment Notes / Project Notes 19

Help access for the Aria software 20

Help system 20 Sample experiments 21

Agilent Technologies

1 Getting Started with the Aria Software

The Aria software

The Aria software features a variety of experiment type options with customized plate and thermal profile setup, and experiment analysis screens that streamline the process of collecting and analyzing data for specific applications. The software capabilities allow you to:

• Quickly set up an experiment using a template or the Import Plate Setup function

• View raw fluorescence data without mathematical correction or calibration factors

• Quantitate the initial template quantity of a DNA target based on a standard curve

• Generate and display normalized relative quantity values on a log(2) fold change chart to assess the effects of an experimental treatment on gene expression levels

• Export any data set directly to Microsoft Excel or to a text file

• View and analyze the data from several experiments together in a single

project using the multiple experiment analysis functions

• Use high resolution melt analysis for SNP genotyping in an Allele Discrimination experiment

15 Agilent Aria Real-Time PCR Program

Getting Started with the Aria Software

Introduction to the Aria software

The software's Home screen provides an introduction to the program and a list of program features.

To open the Home screen: Click the Home icon near the upper right corner of the program window.

Getting Started with the Aria Software 1

Getting Started with the Aria Software

NOTE

NOTE

If you want the program to open to the Home screen, mark the check box near the bottom of the screen labeled Show Home on StartUp.

Agilent Aria Real-Time PCR Program 16

1 Getting Started with the Aria Software

Getting Started with the Aria Software

Introduction

To view the Introduction, click Introduction (near the top right corner of the Home screen). The text on this screen provides an overview of the AriaMx/AriaDx Real- Time PCR System.

Features

To view the Features, click Features (near the top right corner of the Home screen). The text on this screen lists the features of the Aria software.

Minimum PC requirements

Table 1 lists the minimum PC requirements needed to run the Aria

software.

Table 1 Minimum requirements for running the Aria software

Operating system Windows 10 – with regional format set to English (United States)

Supported architectures ×64 (64 bit)

Programs* Microsoft .NET Framework 4.0

Microsoft SQL Server 2012 (required for ET software only)

Runtime components of Microsoft Visual C++ 2010 Libraries

Processor 2 GHz Dual Core Processor

Working memory (RAM) 2 GB (more is recommended)

Hard disk space 40 GB

Display resolution 1024 × 768 (1280 × 1024 is recommended)

* Installers for Microsoft .NET Framework 4.0 and Microsoft SQL Server 2012 are provided

on the Aria Software Download page of the Agilent website. If you do not have the needed Microsoft Visual C++ 2010 components, then the Aria installer will automatically install them to your PC when you initiate installation of the Aria software.

17 Agilent Aria Real-Time PCR Program

Overview of the user interface

Home screen

The Home screen provides an introduction to the AriaMx/AriaDx system and a list of program features. See “Introduction to the Aria software” on page 16 for detailed information on the Home screen.

Menu toolbar

At the top of the program window are the File and Instrument menus.

• The File menu contains commands for opening, closing, saving, and printing experiments and projects.

• The Instrument menu contains commands for connecting to, configuring, exporting data from, and testing the AriaMx/AriaDx instrument.

Getting Started with the Aria Software 1

Overview of the user interface

Tabs

The user interface of the Aria software allows you to open up to 5 experiments at a time (when experiment files are < 1.5 MB), or one project at a time. The program displays each open experiment or project on its own tab, enabling you to quickly switch between them. Click the icon to the right of the tabs to open a new tab. New tabs open to the Getting Started screen.

Left and right panels

When you are working in an experiment or project, the left side of the screen displays the Experiment Area panel, and the right side of the screen displays a panel with tools for the currently open screen (e.g., the Report Configuration panel is displayed on the Generate Report screen). You can hide these panels by clicking the arrow icon next to the panel name. Click the arrow icon again to expand the panel.

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1 Getting Started with the Aria Software

Overview of the user interface

Home/Notifications/Help icons

The top right corner of the program window has 3 icons for quickly accessing the Home screen, viewing any notifications from the program, and opening the Aria help system.

- Click this icon to open the Home screen. Click the icon again to close

the Home screen and return to previous screen.

- Click this icon to open a text box displaying any notifications from the program. When you have unread notifications waiting, the icon is dark blue.

- Click this icon to open the program's help system to the topic that

pertains to the currently displayed screen.

Getting Started screen

When you open a new tab in the program (from the File menu or the icon), it opens to the Getting Started screen. From this screen you can create a new experiment (from scratch or from a template), create a new multiple experiment analysis project, or open an existing experiment or project. See “Overview of the Getting Started screen” on page 43 for more information.

Experiment Notes / Project Notes

The Experiment Notes icon or Project Notes icons appears in the lower left corner of the screen whenever an experiment or project is open. Clicking the icon opens the Experiment Notes or Project Notes text box. Use this text box to type your own notes pertaining to the particular experiment or project. Click Save to save your notes for later reference.

19 Agilent Aria Real-Time PCR Program

Help access for the Aria software

The Aria software contains a help system that provides instructions on setting up, running, and analyzing experiments and creating multiple experiment projects. You can also view video tutorials on the Agilent website that describe how to perform some of the most common tasks in the program.

Help system

From any screen or dialog box, click the help icon ( ) in the upper right corner of the screen to open the help system. The help system automatically opens to the most relevant topic based on where you are in the program.

You can navigate the help system window from the Contents tab or the Search tab.

• On the Contents tab, use the table of contents on the left side of the

window to browse the chapters and topics within the help system.

• On the Search tab, type a search word into the field and click GO to

find the help topics that contain the word. If you type multiple words into the field, the program will list help topics that contain all of the words. Use quotes to search for a complete phrase (e.g., “comparative quantitation”). You can also use the Boolean operators AND and OR to find topics that contain more than one search word (e.g., comparative AND quantitation), or topics that contain any one of multiple search words (e.g., comparative OR quantitation).

Getting Started with the Aria Software 1

Help access for the Aria software

The help system contains the following chapters:

• Getting Started with the Aria Software- Contains help topics to

familiarize new users with the program and provide instructions for getting started

• How- To Help - Provides step- by- step instructions on how to use the

program

• Reference Help - Contains trademark designations and a glossary of

QPCR terms

• Troubleshooting and Support - Contains troubleshooting suggestions

and a directory for contacting a technical support person in your region

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1 Getting Started with the Aria Software

Help access for the Aria software

Sample experiments

The Aria software comes with several sample experiment files that include post- run data. The sample experiments are saved to the folder C:\Users\Public\Public Documents\Agilent Aria\Sample Experiments during installation of the software. The folder includes a sample experiment for each experiment type and subtype for both the AriaMx (*.amxd) and AriaDx (*.adxd) software modes. You open the sample experiments in the Aria software to help familiarize yourself with the experimental setup and graphical displays available for each experiment type.

21 Agilent Aria Real-Time PCR Program

2 Specifying Hardware and Software Settings

Update instrument optics 23 Change the default crosstalk correction settings 25 Set software preferences 28

Agilent Technologies

2 Specifying Hardware and Software Settings

Update instrument optics

Multiple optical modules are available for use with the AriaMx/AriaDx instrument for detection of different dye spectra. In the Supported Optical Configuration dialog box, you can view the optical modules, their currently supported dyes, and the crosstalk correction settings for non- target dyes that could be detected by each module.

Periodically, Agilent may add new supported dyes and optical modules to the AriaMx/AriaDx Real- Time PCR System, and make a new optics configuration file available to you. You can use the Supported Optical Configuration dialog box to load that new file.

To open the Supported Optical Configuration dialog box: At the top of the program window, click Instrument > Optical Configuration.

23 Agilent Aria Real-Time PCR Program

Specifying Hardware and Software Settings 2

Update instrument optics

To update the optical configuration file:

1 Open the Supported Optical Configuration dialog box.

2 Click Import Optical Configuration.

The Open dialog box opens.

3 Browse to the folder of the configuration file that you received from

Agilent. Select the file and click Open. The Supported Optical Configuration dialog box is updated with the new configuration.

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2 Specifying Hardware and Software Settings

Change the default crosstalk correction settings

Multiple optical modules are available for use with the AriaMx/AriaDx instrument for detection of different dye spectra. In the Supported Optical Configuration dialog box, you can view the optical modules and their currently supported dyes, and change the crosstalk correction settings for non- target dyes that could be detected by each module.

To open the Supported Optical Configuration dialog box: At the top of the program window, click Instrument > Optical Configuration.

Crosstalk occurs when emission from one dye is detected by two different optical modules (the target optical module and the spillover optical module). A dye is at risk for crosstalk when its emission spectra overlaps that of another dye that is assigned to a different optical module. The Aria software includes crosstalk correction settings, which can help compensate for crosstalk.

25 Agilent Aria Real-Time PCR Program

Specifying Hardware and Software Settings 2

Change the default crosstalk correction settings

The factory settings for crosstalk correction are the default settings, unless the default settings are changed in the Supported Optical Configuration dialog box. Once changed, the new default crosstalk correction settings are applied to all experiments going forward. Alternatively, you can adjust the crosstalk correction settings for an individual post- run experiment using the tools in the Amplification Plots graph. See “Adjust the crosstalk

correction settings” on page 201 for instructions.

NOTE

The factory settings for crosstalk correction have been optimized to eliminate potential crosstalk for dyes that are part of the default optical configuration. Agilent does not recommend changing the crosstalk correction settings unless you are using a new custom dye and the emission wavelength of that dye could be detected by more than one optical module in the instrument.

To change the default crosstalk correction settings:

1 In the Supported Optical Configuration dialog box, locate the box for

the optical module that is or could be reporting crosstalk fluorescence (i.e., the spillover optical module). The dyes that have the potential to crosstalk with that optical module are listed in that box (e.g., the HEX- JOE dye in the FAM optical module).

2 Change the crosstalk correction setting for the dye by adjusting the

value in the field.

The values in these fields are percentages of the total raw fluorescence. They will be subtracted from the raw fluorescence signal for the optical module when that dye is used as a target.

Crosstalk correction values that differ from the factory settings are noted with an asterisk (*).

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2 Specifying Hardware and Software Settings

Change the default crosstalk correction settings

3 Click OK to apply your changes and close the dialog box.

To reset the crosstalk correction settings back to the factory settings:

1 In the Supported Optical Configuration dialog box, locate the box for

the optical module that you want to reset.

Crosstalk correction values that differ from the factory settings are noted with an asterisk (*).

2 Within that box, click the reset icon next to Crosstalk Correction.

The crosstalk correction values for all dyes listed in the box are reset to the factory values.

3 Click OK to apply your changes and close the dialog box.

NOTE

The Supported Optical Configuration dialog box does not include crosstalk correction settings between the HEX-JOE and Cy3 optical modules because the degree of crosstalk is too significant to be adequately corrected. Agilent does not recommend using HEX-JOE and Cy3 together in a multiplex reaction.

27 Agilent Aria Real-Time PCR Program

Set software preferences

The Preferences dialog box is used for setting your preference on the default file name configuration and for creating and selecting analysis templates.

To open the Preferences dialog box: At the top of the program window, click File > Preferences.

Set default file name configuration

To configure the default file name for experiments and projects:

1 In the Preferences dialog box, make sure File is selected at the top.

Specifying Hardware and Software Settings 2

Set software preferences

2 Next to New Experiment/Project File Naming, click one of the

following options:

• Default - Select this option to use the program's default file naming system. For experiments, the default file name is “Experiment” followed by the next available number (e.g., “Experiment 3"). For projects, the default file name is “Project” followed by the next available number (e.g., “Project 3").

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2 Specifying Hardware and Software Settings

Set software preferences

• Custom - Select this option to configure your own file naming system by selecting which pieces of information to include in the default file name. Using the check boxes, you can select to include the experiment type, the creation date for the experiment or project (in format MM- DD- YYYY, DD- MM- YYYY, or YYYY- MM- DD), and the time that the experiment or project was created.

3 Click OK to close the dialog box and save your changes.

Create and apply analysis templates

To create an analysis template that sets the default analysis settings for experiments:

1 In the Preferences dialog box, select Defaults at the top.

2 Type a name for the new template into the field below Choose analysis

template (and Select OK to apply).

29 Agilent Aria Real-Time PCR Program

Specifying Hardware and Software Settings 2

Set software preferences

3 Select when to apply the analysis template using the check boxes. You

can mark one check box, both check boxes, or neither check box.

• Mark Creating new experiment if you want to apply the analysis template to all newly created experiments going forward.

• Mark Opening post run experiment if you want to apply the analysis template anytime a post run experiment is opened in the program.

• Clear both check boxes if you do not want the analysis template to be automatically applied to experiments.

4 Click Add.

A message box opens asking you to confirm that you want to create the new template. Click OK to continue.

5 Click OK to close the Preferences dialog box.

The program will apply the template to your experiments according to the check box selections made in step 3.

Newly created analysis templates have only default analysis settings. See

“Configure and apply analysis templates” on page 192 for instructions

on configuring the analysis settings in the analysis template.

To select an existing analysis template to be applied to experiments:

1 In the Preferences dialog box, select Defaults at the top.

2 In the field below Choose analysis template (and Select OK to apply),

click the arrowhead to expand the drop- down list. The list contains all of the existing analysis templates.

3 Select a template from the list.

4 Select when to apply the analysis template using the check boxes. You

can mark one check box, both check boxes, or neither check box.

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2 Specifying Hardware and Software Settings

Set software preferences

• Mark Creating new experiment if you want to apply the analysis

template to all newly created experiments going forward.

• Mark Opening post run experiment if you want to apply the

analysis template anytime a post run experiment is opened in the program.

• Clear both check boxes if you do not want the analysis template to

be automatically applied to experiments.

5 Click OK to close the Preferences dialog box. The program will apply

the template to your experiments according to the check box selections made in step 3. See “Configure and apply analysis templates” on page 192 for instructions on configuring the analysis settings in the analysis template.

To delete an existing analysis template:

1 In the Preferences dialog box, select Defaults at the top.

2 In the field below Choose analysis template (and Select OK to apply),

click the arrowhead to expand the drop- down list. The list contains all of the existing analysis templates.

3 Select a template from the list.

4 Click Delete.

5 In the message box that opens, click OK to confirm that you want to

delete the selected template.

6 If desired, select a different template from the list, or create a new

template.

7 When finished making changes, click OK to close the Preferences dialog

box.

31 Agilent Aria Real-Time PCR Program

Performing Hardware and Software Tests and HRM Calibrations

Run an instrument qualification test 33

Run the test 33

About the Qualification Test graphical data 34 Perform an Installation Qualification test 36 Run an HRM calibration plate (HCP) 38

Prepare the plate 38 Run an HCP 39

If the HCP fails 40

Agilent Technologies

3 Performing Hardware and Software Tests and HRM Calibrations

Run an instrument qualification test

Running a qualification test is one way to test for instrument errors. You perform the test using a qualification test plate that must be purchased separately from Agilent (part number 5190- 7708). The wells of the test plate are pre- filled with the QPCR reagent mixture needed to run the test.

Run the test

Running a qualification test requires preparing the plate, running the experiment on the AriaMx/AriaDx instrument, and checking the results.

To run a qualification test:

1 Prepare the qualification test plate according to the instructions that

came with the plate.

2 At the top of the program window, click Instrument > Qualification

Test.

A new tab opens for the Qualification Test experiment. The experiment opens to the Thermal Profile screen.

3 Click Run.

The Instrument Explorer dialog box opens.

4 Locate the instrument that you will be using for the run and click Send

Config.

• If the instrument is not listed, see “Add instruments to your

network” on page 162 for instructions on searching for and adding

instruments.

• If this is the first time you have connected to an instrument since last launching the Aria software, the Login dialog box opens. Select your Username from the drop- down list, type your login password into the Password field, and click Login. To log in with a different user account, right- click on the instrument name and click Log off current user. You can then log in using the desired user account

A message box opens notifying you that you must save the experiment before you can connect to the instrument.

5 Click Save in the message box.

The Save As dialog box opens.

33 Agilent Aria Real-Time PCR Program

Performing Hardware and Software Tests and HRM Calibrations 3

Run an instrument qualification test

6 Select a folder for the experiment and type a name into the File name

field (or use the default). Click Save.

7 Take the prepared qualification test plate over to the instrument and

load it into the thermal block.

8 On the instrument touchscreen, open the primed experiment to the

Thermal Profile screen and press Run Experiment.

The instrument starts running the qualification test experiment.

9 Return to the PC program. The program directs you to the Raw Data

Plots screen, where you can monitor the progress of the run. See

“Monitor a run” on page 168.

10 At the end of the run, save the qualification test experiment to your PC

(see “Retrieve run data from the instrument” on page 173 for instructions). Open the experiment in the Aria software on your PC.

11 Click Graphical Displays in the Experiment Area panel on the left side

of the screen. (See “About the Qualification Test graphical data” on page 34 for more information about the graphs on this screen.)

12 Check the panel on the right side of the screen to determine if the

qualification test passed.

• If it passed, the panel displays Results Pass.

• If it failed, the panel displays Results Check.

If your qualification test failed, contact Agilent Technical Support for help with troubleshooting. See “Contact Agilent Technical Support”.

About the Qualification Test graphical data

For a qualification test, the Graphical Displays screen includes graphs for the Amplification Plots, the Standard Curve, and the Population Distribution.

The Population Distribution graph is unique to Qualification Test experiments. For each row of Unknown wells on the plate, this graph plots the number of initial template copies calculated for that row against the plate's column number. Because all wells in rows A, B, and C are replicates and all wells in rows F, G, and H are replicates, the distribution of the plot lines indicate the variability in initial template calculations across the thermal block within the two populations (the ABC population and the FGH population). In order to pass the qualification test, the

Agilent Aria Real-Time PCR Program 34

3 Performing Hardware and Software Tests and HRM Calibrations

Run an instrument qualification test

program requires that both populations be at least 98.5% distinct after outlier wells are culled. The percent distinct for the Population Distribution graph is displayed in the panel on the right side of the Graphical Displays screen (next to % Distinct). Note that outlier wells are plotted on the Population Distribution graph with a red x.

If your qualification test failed, Agilent's Technical Support staff may use the graphical data to help troubleshoot the cause of the failure.

35 Agilent Aria Real-Time PCR Program

Performing Hardware and Software Tests and HRM Calibrations 3

Perform an Installation Qualification test

You can run an Installation Qualification (IQ) test to determine if the Aria software is properly installed on your system. The IQ test is comprised of three separate tests:

• GUI Software Installation Qualification: Verifies the integrity of the files and folders that are created as part of Aria software installation

• Permissions test: Verifies that the current user has full access to the software's default file path for experiment files

• Connectivity test: Tests the communication between software and instrument via FTP or TCP protocol service (if the firmware version is determined to be outdated, a message box opens prompting you to upgrade the firmware)

To perform the IQ test:

1 Click File > Qualifications. The Qualifications dialog box opens.

Perform an Installation Qualification test

Agilent Aria Real-Time PCR Program 36

3 Performing Hardware and Software Tests and HRM Calibrations

Perform an Installation Qualification test

2 Next to Report is the file path and file name of the report that the

program generates at the end of the test. To select a different folder or file name for the report, click Change. Then, in the Save As dialog box, select a different folder and/or file name.

3 Click Start to start the test.

The program displays the status of the test in the Test Log area of the dialog box. When finished, the program updates the Status column in the dialog box and displays the overall result (Passed or Failed). If the test failed, contact Agilent Technical Support for assistance.

4 Click Open Report to open the PDF report generated by the program.

5 To run the test again, click Reset to clear the results of the previous

test.

NOTE

The Operational option on the Qualifications dialog box has tools to run an Operational Qualification (OQ) test. However, the Operational option is only available to Agilent Service Engineers with an appropriate password.

37 Agilent Aria Real-Time PCR Program

Performing Hardware and Software Tests and HRM Calibrations 3

Run an HRM calibration plate (HCP)

For experiments that include a high resolution melt (HRM) segment, before you can analyze the HRM data on a Difference Plots graph, you must associate the experiment with an HRM calibration plate (HCP) that was run on the same instrument. The purpose of the HCP is to calculate an offset temperature for each well in order to help normalize well- to- well temperature variations. See “About HRM calibration plates” on page 67 for more information.

Run an HRM calibration plate (HCP)

NOTE

Prepare the plate

Use the Agilent HRM Calibration Plate

Agilent offers a HRM Calibration Plate that you can use for HRM calibration (Agilent part number 5190- 7701). The plate comes pre- aliquoted with a master mix containing EvaGreen dye. See the plate's user manual for more information.

Visit www.agilent.com/genomics for ordering information on the HRM Calibration Plate.

Prepare the plate with your own reagents

Alternatively, you can prepare your own reagent mixture to be used in the HCP run.

The 1x mixture needs to contain a purified amplicon at a concentration of

0.1- 0.3 mM. Ideally, the amplicon has a melting temperature (Tm) close to

that of the amplicon that you will be analyzing in your Allele Discrimination experiment. The 1x mixture also needs to contain the same DNA binding dye, at the same concentration, that you use in the QPCR reactions for the Allele Discrimination experiment.

A separate HRM software license is required in order to associate an experiment with an HCP. To purchase a software license, contact your Agilent Sales representative. The HRM license option is only available for the AriaMx system. The license is not supported for the AriaDx system.

Agilent Aria Real-Time PCR Program 38

3 Performing Hardware and Software Tests and HRM Calibrations

Run an HRM calibration plate (HCP)

Instead of using a purified amplicon in your reagent mixture, you can use a template, primers, and polymerase to produce the amplicon during the HCP run. If you choose this approach, you need to add an amplification segment to the default HCP thermal profile.

Note that the Aria calibration algorithms were tested and optimized using the Agilent HRM Calibration Plate. Using your own HCP reagent mixture may impact results.

Run an HCP

All HCP experiments must be set up and run directly from the instrument (you cannot set up an HCP experiment from the Aria program on your PC).

To run an HCP experiment:

1 On the instrument Home screen, press the HRM Calibration icon.

2 On the subsequent screen, press Open Default Experiment.

The default HCP experiment opens to the Plate Setup screen. All wells are set to the Unknown well type and the SYBR dye is selected for target detection in all wells

The EvaGreen dye used in the Agilent HCP kit is detectable with the FAM/SYBR optic module.

3 Navigate to the Thermal Profile screen and press Run Experiment.

A message box opens on the touchscreen prompting you to save the experiment. Press OK in the message box to save the experiment to the HCP folder.

4 Select a file name for the experiment and press Save.

The instrument starts running the experiment.

5 After the run, a message box opens on the screen notifying you if the

HCP passed or failed.

• If it passed, copy the post- run HCP experiment file to your PC (see

“Retrieve run data from the instrument” on page 173 for

instructions). You can then use the HCP to calibrate HRM data from an experiment. See “Assign an HRM calibration plate” on page 183 for instructions.

39 Agilent Aria Real-Time PCR Program

• If it failed, you cannot use the HCP to calibrate HRM data from an

If the HCP fails

If the HCP fails the system's quality check, the touchscreen displays a message box at the end of the HCP run notifying you of the failure. You will also see a warning icon (as shown below) if you open the experiment in the Aria program on your PC.

Possible causes of a failed plate include pipetting errors during set up of the plate and amplicon degradation. If you find your HCP runs repeatedly fail, try setting up the plate using the Agilent HRM Calibration Plate. If problems persist, contact Agilent Technical Support (see “Contact Agilent

Technical Support” on page 370). Note that you cannot associate a failed

HCP with an experiment.

Performing Hardware and Software Tests and HRM Calibrations 3

Run an HRM calibration plate (HCP)

experiment. See “If the HCP fails”, below, for information on failed HCPs.

Agilent Aria Real-Time PCR Program 40

3 Performing Hardware and Software Tests and HRM Calibrations

Run an HRM calibration plate (HCP)

41 Agilent Aria Real-Time PCR Program

4 Creating/Opening an Experiment

Overview of the Getting Started screen 43

Tools available on the Getting Started screen 44 About the Aria file types 45

Quick Start Protocol 46

How to create, set up, run, analyze, and generate reports for an

experiment 46

Create a new experiment 49

Create an experiment based on experiment type 49 Create an experiment from a template 50

Create an experiment from a LIMS data file 51 Open an existing experiment 53 Save a copy of an existing experiment 54 Create a template from an existing experiment 55 Convert an experiment to a new experiment type 56

Agilent Technologies

4 Creating/Opening an Experiment

Overview of the Getting Started screen

The tools on the Getting Started screen allow you to create a new experiment (from scratch or from a template), create a new multiple experiment analysis project, or open an existing experiment or project.

To open the Getting Started screen: At the top of the program window, click File > New. Alternatively, click the icon to open a new tab in the program. The new tab opens to the Getting Started screen.

43 Agilent Aria Real-Time PCR Program

Creating/Opening an Experiment 4

Overview of the Getting Started screen

Tools available on the Getting Started screen

The content in the center of the screen changes depending on which option is selected in the panel on the left. These options are described in the table below

Option Description

New Experiment

Experiment Types Click this option to create a new experiment based on the desired experiment type.

The center of the screen displays the experiment types for selection.

My Templates Click this option to create a new experiment from a template. The center of the

screen displays the template files in the Experiments Templates folder that is created during program installation.

From LIMS file... Click this option to create a new experiment from a LIMS data file that specifies the

plate setup and, optionally, the thermal profile. The center of the screen displays the Import From LIMS Data File wizard.

New Project

Multiple Experiment Analysis

Saved

Recently Opened Click this option to open an existing experiment or project that you recently

Browse Click this option to open an existing experiment or project by browsing to a desired

Links at the bottom of the screen

License Click this link to open a message box about licensing. In the message box, click

Contact Support Click this link to create a new email message directed to the Agilent Technical

Agilent Aria Real-Time PCR Program 44

Click this option to create a new project for analyzing and comparing multiple post-run experiments. The center of the screen displays tools for adding experiments to the new project.

accessed. The center of the screen displays a list of experiments that you have recently opened.

folder.

License Agreement to view the full text of the Aria software license agreement.

number of the software.

Support group.

4 Creating/Opening an Experiment

Overview of the Getting Started screen

About the Aria file types

Three different files types can be created in the Aria program: an experiment file, a protocol template file, and a project file. These file types are summarized below.

Experiment Files (*.amxd or *.adxd)

In the Aria software, experiments of all types (Quantitative PCR, Comparative Quantitation, Allele Discrimination, and User Defined) are saved as experiment files. When an experiment file is open, the program includes screens for defining the wells of the experimental plate, setting up the thermal profile, running the experiment, and analyzing the results of that run. AriaMx experiment files are given the extension amxd, and AriaDx experiment files are given the extension adxd.

Template Files (*.amxt or *.adxt)

In addition to saving an experiment as an experiment file, the plate setup and thermal profile of an experiment can be saved as a template file. Creating a new experiment from a template file allows you to quickly set up new experiments that require a similar plate setup or thermal profile. AriaMx template files are given the extension amxt, and AriaDx template files are given the extension adxt.

Project Files (*.amxp or *.adxp)

Multiple experiment analysis projects are saved as project files. Up to 8 post- run experiment files (of the same experiment type) can be added to a project, enabling you to analyze the results side by side or combine results across experiments. AriaMx project files are given the extension amxp, and AriaDx project files are given the extension adxp.

45 Agilent Aria Real-Time PCR Program

Quick Start Protocol

How to create, set up, run, analyze, and generate reports for an experiment

1. Create the experiment

• Open a new tab. On the Getting Started screen, under New Experiment, click Experiment Types (to create a new experiment by

the experiment type) or My Templates (to create a new experiment from a template).

2. Set up the plate

• After creating the experiment, the experiment opens to the Plate Setup screen.

• Assign the plate properties based on experiment type, including assigning well types, replicate numbers, and dyes/targets. See the topics below for instructions specific to each experiment type.

“Assign plate properties for a Quantitative PCR DNA Binding Dye experiment” on page 86

“Assign plate properties for a Quantitative PCR Fluorescence Probe experiment” on page 94

“Assign plate properties for a Comparative Quantitation experiment”

on page 102

“Assign plate properties for an Allele Discrimination DNA Binding Dye experiment” on page 113

“Assign plate properties for an Allele Discrimination Fluorescence Probe experiment” on page 122

“Assign plate properties for a User Defined experiment” on page 131

Creating/Opening an Experiment 4

Quick Start Protocol

3. Set up the thermal profile

• Navigate to the Thermal Profile screen. (Click Thermal Profile in the Experiment Area panel on the left side of the screen.)

• Edit the thermal profile as desired, or use the default/template thermal profile.

Agilent Aria Real-Time PCR Program 46

4 Creating/Opening an Experiment

Quick Start Protocol

4. Run the experiment

• From the Thermal Profile screen, click Run. In the Instrument Explorer dialog box that opens, locate the instrument and click Send

Config.

• Load your reaction plate into the instrument's thermal block. On the

instrument touchscreen, open the primed experiment to the Thermal Profile screen and press Run.

• If desired, monitor the progress of the run from your PC.

5. Set the analysis criteria

• When the run is finished, navigate to the Analysis Criteria screen. (Click Analysis Criteria in the Experiment Area panel on the left side of the screen.)

• Select the wells, well types, and targets to include in the analysis, specify the treatment of replicate wells, and select the data collection marker to use for analysis. If your experiment included a high resolution melt segment (HRM), assign an HRM calibration plate to the experiment.

6. Analyze the data

• Navigate to the Graphical Displays screen. (Click Graphical Displays in the Experiment Area panel on the left side of the screen.)

• View the results of the analysis and customize analysis settings for individual graphs. See the topics below for instructions specific to each graph.

“View the Amplification Plots” on page 197

“View the Melt Curve - Raw/Derivative Curve” on page 212

“View the Melt Curve - Difference Plots” on page 218

“View the Standard Curve” on page 225

“View the Relative Quantity” on page 230

“View the Allele Determination graph” on page 235

47 Agilent Aria Real-Time PCR Program

Creating/Opening an Experiment 4

Quick Start Protocol

7. Export the results

• To generate a report of the results, navigate to the Generate Report screen (click Generate Report in the Experiment Area panel on the left side of the screen). Configure and create the report according to your selections.

• To export numerical data from the experiment, navigate to the Export Data screen (click Export Data in the Experiment Area panel on the left side of the screen). Select the file type and information you want to export.

Agilent Aria Real-Time PCR Program 48

4 Creating/Opening an Experiment

Create a new experiment

The Getting Started screen allows you to create a new experiment. To create the new experiment from scratch, you start by selecting the experiment type. To create the new experiment from a template or LIMS data file, you start by selecting the template or LIMS data file that you want to use. Once created, you can edit the plate setup and thermal profile for the new experiment.

To open the Getting Started screen: At the top of the program window, click File > New, or click the icon, to open a new tab in the program. The new tab opens to the Getting Started screen.

Create an experiment based on experiment type

When you create a new experiment based on experiment type, your selection of the experiment type determines some of the setup options and analysis outputs. The thermal profile of the new experiment is set to the default for the chosen experiment type. The plate setup of the experiment is blank, but the available well types and other well configuration tools on the Plate Setup screen are specific to the experiment type.

To create an experiment based on experiment type:

1 Open the Getting Started screen.

2 Under New Experiment, click Experiment Types.

The center of the screen displays all the available experiment types. See

“Overview of Experiment Types” on page 59.

3 Click the desired experiment type to select it.

4 In the Experiment Name field, type a name for the new experiment,

then click Create.

The program creates the new experiment and opens the experiment to the Plate Setup screen.

NOTE

49 Agilent Aria Real-Time PCR Program

At step 3, you can double-click the desired experiment type to select it with the default experiment name. The program creates the new experiment and opens the experiment to the Plate Setup screen (or to the Thermal Profile screen for User Defined experiments).

Create an experiment from a template

When you create a new experiment from a template, the experiment has the plate setup and thermal profile of the selected template. After you create the experiment, you can edit the plate setup and thermal profile as desired.

The Aria software comes with three sample template files. The templates are available in the folder C:\Users\Public\Public Documents\Agilent Aria\Experiment Templates.

To create an experiment from a template:

1 Open the Getting Started screen.

2 Under New Experiment, click My Templates.

The center of the screen displays the template files in the default template folder.

Creating/Opening an Experiment 4

Create an experiment from a template

NOTE

You can toggle between displaying the templates in list format and tile format by clicking the icons in top right corner.

= List view icon

= Tile view icon

3 In the Experiment Name field, type a name for the new experiment.

4 Select the desired template and create the experiment.

• If the template is in the default folder, click directly on the template to select it and then click Create (or double- click directly on the template). The program creates the new experiment and opens the experiment to the Plate Setup screen.

• If the template is not in the currently selected folder, click the Browse to Template icon (shown below) to open the browser window. Browse to the folder of the desired template file. Select the file and click Open. The program creates the new experiment and opens the experiment to the Plate Setup screen.

Agilent Aria Real-Time PCR Program 50

4 Creating/Opening an Experiment

Create an experiment from a template

Create an experiment from a LIMS data file

To create a new experiment from a LIMS data file, you must provide a valid file. The file may be generated by exporting a post- run Aria experiment to a LIMS data file (see “Export to a LIMS data file” on page 262), by setting up a text file in the necessary Aria- supported LIMS data file format, or by a LIMS software program. See “LIMS File Format

for Aria Software” on page 361 for information on format requirements for

LIMS data files used to create new experiments in the Aria program. After you import the file and create the experiment, you can edit the plate setup and thermal profile as desired from the Plate Setup and Thermal Profile screens.

The Aria software comes with sample LIMS data files (text files and CSV files). The files are available in the folder C:\Users\Public\Public

Documents\Agilent Aria\Sample LIMS Import Files.

To create an experiment from a LIMS data file:

1 Open the Getting Started screen.

2 Under New Experiment, click From LIMS file....

The center of the screen displays the Import From LIMS Data File wizard.

3 In the Filename field under LIMS data file, type the file path for the

LIMS data file, or click Browse to browse to and select the LIMS data file. The file type can be a text file (*.txt) or a CSV file (*.csv).

The program populates the fields in the Experiment setup and Thermal profile setup areas of the LIMS Data File wizard using the available information from the selected file. Note that the file may not include all experiment details.

4 (Optional) Edit the information in the Experiment setup fields as

desired.

• In the Name field, type a name for the experiment. If the imported

LIMS data file specified the experiment name, then the field is populated with that name.

• In the Type drop- down list, select an experiment type. If the

imported LIMS data file specified the experiment type, then the drop- down list is set to that selection. See “Overview of Experiment

Types” on page 59 for descriptions of the Aria experiment type

options.

51 Agilent Aria Real-Time PCR Program

Creating/Opening an Experiment 4

Create an experiment from a template

• In the Notes field, type any experiment notes that you want

associated with the new experiment. If the imported LIMS data file included experiment notes, then the field is populated with those notes.

5 Click Next.

The screen displays the plate setup information for the experiment.

6 (Optional) Edit the Reference Dye selection and other target information

as permitted for the experiment type.

7 Click Finish.

The program creates the new experiment and opens the experiment to the Plate Setup screen. A message box opens confirming that the experiment has been successfully imported. Click OK to close the message box.

Agilent Aria Real-Time PCR Program 52

4 Creating/Opening an Experiment

Open an existing experiment

The program allows you to open up to 5 experiments at a time, or one project at a time. The program displays each open experiment or project on its own tab.

To open an experiment in a new tab:

1 Click the icon to the right of the tabs to open a new tab.

The new tab opens to the Getting Started screen.

2 Click one of the options under Saved:

• To open an existing experiment that you recently accessed, click Recently Opened. The center of the screen displays a list of

experiments and projects that you have recently opened. Double- click the experiment you want to open. The program opens the experiment to the Plate Setup screen.

• To browse to the folder of the experiment, click Browse. The Open dialog box opens. Browse to the folder location of the experiment. Select the experiment and click Open. The dialog box closes and the program opens the experiment to the Plate Setup screen.

To open an experiment in an already open tab:

1 From the toolbar, click File > Open.

The Open dialog box opens. If an experiment or project is currently open in the tab, the program closes that experiment or project, and prompts you to save any changes.

2 Browse to the folder location of the experiment. Select the experiment

and click Open.

The dialog box closes and the program opens the experiment to the Plate Setup screen.

53 Agilent Aria Real-Time PCR Program

Save a copy of an existing experiment

You can use the Save As command to copy the open experiment and save it with a new experiment name.

To copy an existing experiment using the Save As command:

1 Open the existing experiment that you want to copy.

2 Click File > Save As.

The Save As dialog box opens.

3 Select a folder for the new experiment and type a name into the file

name field.

4 Click Save.

The program saves the open experiment under the new name.

Creating/Opening an Experiment 4

Save a copy of an existing experiment

Agilent Aria Real-Time PCR Program 54

4 Creating/Opening an Experiment

Create a template from an existing experiment

You can use the Save As Template command to create a template file based on the plate setup and thermal profile of an existing experiment. You can later use the template to quickly create new experiments. See

“Create an experiment from a template” on page 50.

To create a template from an existing experiment using the Save As Template command:

1 Open the existing experiment that you want to create a template from.

2 Click File > Save As Template.

The Save As dialog box opens. If you are running the AriaMx mode of the software, the file type set to AriaMx Template Files (*.amxt). If you are running the AriaDx mode of the software, the file type set to AriaDx Template Files (*.adxt).

3 Select a folder for the new template and type a name into the file name

field.

4 Click Save.

The dialog box closes and the program saves the new template file (*.amxt or *.adxt) to the designated folder.

55 Agilent Aria Real-Time PCR Program

Convert an experiment to a new experiment type

The Convert Experiment Type command can convert a post- run experiment into another experiment type. This command is useful when the experiment was set up as one type and the data needs to be re- analyzed as a different experiment type. The program applies the analysis algorithms and display options based on the new experiment type.

To convert an experiment to a new experiment type:

1 Open the existing post- run experiment that you want to convert.

2 Click File > Convert Experiment Type, and in the sub-menu, select the

new experiment type.

A message box opens notifying you that the conversion was successful and that some well types may have changed.

3 Click OK to close the message box.

The program creates a new experiment file for the converted experiment and opens the experiment to the Plate Setup screen. By default, the new experiment has the same name as the parent experiment with the word “Converted” added at the beginning.

4 Click File > Save As.

The Save As dialog box opens.

5 Select a folder for the new experiment. Type a name into the file name

field or use the default file name.

6 Click Save.

The program saves the new experiment to the designated folder.

Creating/Opening an Experiment 4

Agilent Aria Real-Time PCR Program 56

4 Creating/Opening an Experiment

Convert an experiment to a new experiment type

57 Agilent Aria Real-Time PCR Program

Selecting an Experiment Type

Overview of Experiment Types 59

Quantitative PCR 59 Comparative Quantitation 59 Allele Discrimination - Fluorescence Probes 60 Allele Discrimination - DNA Binding Dye with High-Resolution

Melt 60 User Defined 60

The Quantitative PCR Experiment Type 61

Multiplexing quantitative PCR experiments 61 Well types for Quantitative PCR experiments 62

The Comparative Quantitation Experiment Type 63

Normalizing chance variations in target levels 63 Determining amplification efficiencies for the targets of interest and

normalizer targets 64 Including biological replicates in comparative quantitation 65 Well types for Comparative Quantitation experiments 66

The Allele Discrimination - DNA Binding Dye Experiment Type 67

About HRM calibration plates 67 Well types for Allele Discrimination - DNA Binding Dye

experiments 68

The Allele Discrimination - Fluorescence Probe Experiment Type 69

Well types for Allele Discrimination - Fluorescence Probe

experiments 70

The User Defined Experiment Type 71

Agilent Technologies

5 Selecting an Experiment Type

Overview of Experiment Types

The Aria program offers a variety of experiment types. Each experiment type was designed for a specific application and has its own unique options for experimental setup and analysis that are specialized for that application. The Aria experiment types are summarized below.

Quantitative PCR

The Quantitative PCR experiment type is the best choice when you need to determine the exact quantity of a particular DNA target in the experimental template samples. This experiment type uses a standard curve produced with samples of known template quantity to derive the initial quantity of the target in the experimental sample.For more information, see “The Quantitative PCR Experiment Type” on page 61.

The program offers two sub- types for the Quantitative PCR experiment type: DNA Binding Dye Including Standard Melt and Fluorescence Probe. These sub- types differ by the type of chemistry used to detect the PCR products. In the DNA Binding Dye Including Standard Melt sub- type, detection is based on signal from a double- stranded DNA binding dye, e.g., SYBR Green, and the default thermal profile includes a melt curve. In the Fluorescence Probe sub- type, a target- specific probe, e.g., a TaqMan probe, is used for target detection.

Comparative Quantitation

The Comparative Quantitation experiment type is best used for comparing levels of RNA or DNA across samples when you do not require information about the absolute amount of target. Most common is the comparison of amounts of mRNA in treated versus untreated, or normal versus diseased cells or tissues. The program will ask you to identify which wells contain the control sample (called the “calibrator”) and which wells contain the associated experimental sample (called the “unknown”). For accurate results, you need to normalize the data to the quantity of a target gene (called a “normalizer”) that is known to be unaffected by the experimental conditions, such as a housekeeping gene. For more information, see “The Comparative Quantitation Experiment Type” on page 63.

59 Agilent Aria Real-Time PCR Program

Selecting an Experiment Type 5

Overview of Experiment Types

Allele Discrimination - Fluorescence Probes

The Allele Discrimination experiment type is used to discriminate between two alleles in a DNA sample. For more information, see “The Allele

Discrimination - Fluorescence Probe Experiment Type” on page 69.

The program offers two sub- types for the Allele Discrimination experiment type. In the sub- type Fluorescence Probe, you use two fluorogenic probes labeled with different dyes to discriminate between two alleles in a DNA sample. For example, if amplification in an unknown DNA sample is detected for the dye identifying the wild- type allele but not for the dye identifying a mutant allele, the sample can be designated as wild- type homozygous.

Allele Discrimination - DNA Binding Dye with High-Resolution Melt

The Allele Discrimination experiment type is used to discriminate between two alleles in a DNA sample. For more information, see “The Allele

Discrimination - DNA Binding Dye Experiment Type” on page 67.

User Defined

The User Defined experiment type provides the greatest flexibility in setup and analysis of an experiment. All the well types and other plate setup options that are available across the other experiment types are available on the Plate Setup screen in a User Defined experiment. Similarly, on the Thermal Profile screen, you can add any type of segment to the thermal profile, and on the Graphical Displays screen, you can view the results for any of the experiment type- specific graphs.

Agilent Aria Real-Time PCR Program 60

5 Selecting an Experiment Type

The Quantitative PCR Experiment Type

In Quantitative PCR experiments, the instrument detects the fluorescence of one or more dyes or fluorophores during each cycle of the thermal cycling process and a fluorescence value is reported for each dye/fluorophore at each cycle. Generally, you want the instrument to acquire fluorescence readings during the annealing stage of thermal cycling.

You can quantify the initial copy numbers of RNA or DNA targets based on quantification cycle (Cq) determinations. The Cq is defined as the cycle at which a statistically- significant increase in fluorescence is first detected. The threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background. The Aria program offers both automatic and manual methods for determination of the threshold fluorescence level that is used to determine Cq values.

Typical Quantitative PCR experiments use a standard curve to quantitate the amount of target present in an experimental sample (called the “unknown” sample since the quantity of the target is unknown). In this method, you set up the plate to amplify a series of standards to generate a standard curve that relates initial template quantity to Cq. You generate the standards by serial dilution of a template sample that contains a known quantity of the target under investigation. The program then uses the standard curve to derive the initial template quantity of this target in the unknown samples based on their Cq values.

Multiplexing quantitative PCR experiments

The instrument records fluorescence readings for each sample on all five optical modules. This allows you to use multiplex PCR for quantitation of multiple targets in the same well by using spectrally- distinct dyes to detect each target. The Aria program reports each target in each well separately on amplification plots and other graphical results displays.

61 Agilent Aria Real-Time PCR Program

Well types for Quantitative PCR experiments

Well Type Description

Unknown Contains a complete reaction mixture including a test template that

contains an unknown amount of the target-of-interest.

Buffer Contains only buffer; used to monitor the background fluorescence

attributable to the buffer.

NAC No amplification control; contains all reaction components except DNA

polymerase.

NTC No template control; contains all reaction components except the

template nucleic acid.

This well type is useful for detecting amplicon contamination.

Standard Contains a complete reaction mixture including a known concentration

of the target-of-interest.

This well type is used to generate a standard curve, which is then used to relate the quantification cycle (Cq) to initial template quantity in Unknown wells and calculate the amplification efficiency.

No RT No reverse transcriptase control; contains all QRT-PCR reaction

components except reverse transcriptase.

In one-step RT-PCR assays, this control is useful for assessing levels of genomic DNA carryover that may contribute to fluorescence increase in the sample.

Selecting an Experiment Type 5

The Quantitative PCR Experiment Type

Agilent Aria Real-Time PCR Program 62

5 Selecting an Experiment Type

The Comparative Quantitation Experiment Type

The Comparative Quantitation experiment type provides an efficient method for comparing levels of RNA or DNA across samples when you do not require information about the absolute amount of target in any sample. The most common application is the comparison of amounts of mRNA in treated versus untreated, or normal versus diseased cells or tissues.

For many gene expression studies, your experiments do not require you to determine the absolute amount of a target in a particular sample; evidence of a relative increase or decrease in expression, compared to a sample of reference, is sufficient. The sample of reference is referred to as the calibrator. For example, in a study in which a large number of compounds are screened for the ability to induce the expression of a certain set of genes in HeLa cells, the calibrator might be a nucleic acid sample isolated from an untreated HeLa cell culture. In a study involving the expression of a cancer marker gene, the calibrator might be a nucleic acid sample isolated from the normal, non- diseased part of the organ, whereas the test samples (referred to in the program as Unknowns) are nucleic acids isolated from the diseased tissue of the same patient. The expression level of the target- of- interest (i.e., the gene you are studying) in the calibrator is defined as 1× (or 1.0). Expression levels in all unknown samples are reported as a fold difference relative to this calibrator benchmark.

Normalizing chance variations in target levels

The quantity of a target- of- interest present across a set of independently- isolated samples is subject to many variables such as sample- to- sample differences in total amount of input nucleic acid and differences in the efficiency of RNA extraction or reverse transcription. You can include a normalizer target in the assay to reduce the effect of these spurious variations that do not reflect true differences in target abundance as a result of experimental treatment. Any gene with little to no variance in expression due to the experimental treatment can serve as a normalizer. (Commonly- used examples include housekeeping genes such as GAPDH, cyclophilin, GUS, TFIID, or 18S, or 28S ribosomal RNA.) The abundance of the normalizer and the target- of- interest should be similar.

In a typical Comparative Quantitation experiment, you would set up the wells containing the calibrator sample to run alongside a variety of

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Selecting an Experiment Type 5

The Comparative Quantitation Experiment Type

unknown samples to test the effect of some variable on the expression level of one or more genes of interest. You can set up the reactions amplify the normalizer target in the same well as the target- of- interest (using multiplexing) or set up the reactions to amplify the normalizer and the target- of- interest in different wells.

During analysis, the program automatically adjusts the levels of the target- of- interest in both Unknown and Calibrator wells to compensate for differences in the levels of the normalizer. The program then compares the normalized value for each unknown sample to the normalized calibrator value, and reports the relative quantity for each unknown. (The expression level of the target- of- interest in the Calibrator wells is set to 1.0.)

Establish the amplification efficiencies of the normalizer target and the target- of- interest before you use a particular normalizer in a Comparative Quantitation experiment.

See the following section (“Determining amplification efficiencies for the

targets of interest and normalizer targets”) for more information.

Determining amplification efficiencies for the targets of interest and normalizer targets

In developing a Comparative Quantitation experiment, it is important that the amplification efficiencies of the target- of- interest and the normalizer target are reproducible and, ideally, very similar. To measure the amplification efficiency for each target, generate a standard curve in a Quantitative PCR experiment. When running a standard curve for the sole purpose of determining the amplification efficiency for a particular target, it is not necessary to know the exact quantity of your targets. Instead, you can use serially diluted template as the standard samples and assign the standard quantities in Plate Setup using the “relative” unit designation. The program then analyzes the standard curve data and calculates the amplification efficiency from the slope of the curve. (See “View the

R- squared values, slopes, and amplification efficiencies” on page 227 for

more information on deriving amplification efficiencies from standard curves.)

If differences in amplification efficiency exist

In an idealized Comparative Quantitation experiment, the amplification efficiencies for the target- of- interest and normalizer target must be

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5 Selecting an Experiment Type

The Comparative Quantitation Experiment Type

identical in order to allow a direct correction of target levels across samples. However, if you find from your standard curves that the target- of- interest and normalizer have different amplification efficiencies, the program allows you to compensate for this difference using the settings under Amplification Efficiencies in the Graphical Displays screen. To access these settings, expand the menu in the panel on the right side of the Graphic Displays screen. (See “Enter the amplification efficiencies

for the targets ” on page 233 for detailed instructions.)

Including biological replicates in comparative quantitation

In QPCR, biological replicates are template samples that were isolated independently but from biologically- identical sources (sources that are genetically identical are of the same cell type and were treated identically during experimentation). For example, two samples of cDNA that were isolated from the same tissue source in two different mice that were exposed to identical conditions and have the same genotype would be biological replicates. Biological replicates help you determine the level of variability in gene expression for your specific experiment that is due to uncontrolled biological variation from sample to sample.

When setting up the plate for a Comparative Quantitation experiment, you may designate two or more samples as biological replicates while assigning the sample names. Samples that are biological replicates are assigned the same sample name but have different biological replicate ID numbers. During analysis, the program treats biological replicates independently as different samples.

If the experiment includes multiple biological replicates of the calibrator sample, you can designate only one of the samples within the set of replicates as the calibrator. You can change the assignment of the calibrator after the run if you want to re- analyze the results using a different calibrator designation.

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Selecting an Experiment Type 5

The Comparative Quantitation Experiment Type

Well types for Comparative Quantitation experiments

Well Type Description

Unknown Contains a complete reaction mixture including a test template that

contains an unknown amount of the target-of-interest.

Calibrator Contains a complete reaction mixture including an unknown amount of

the target-of-interest.

The level of a target-of-interest in the calibrator wells is set to 1.0 for comparison to the relative quantities in unknown samples.

NTC No template control; contains all reaction components except the

template nucleic acid.

Standard Contains a complete reaction mixture including a known concentration

of the target-of-interest.

No RT No reverse transcriptase control; contains all QRT-PCR reaction

components except reverse transcriptase.

In one-step RT-PCR assays, this control is useful for assessing levels of genomic DNA carryover that may contribute to fluorescence increase in the sample.

NAC No amplification control; contains all reaction components except DNA

polymerase.

Buffer Contains only buffer; used to monitor the background fluorescence

attributable to the buffer.

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5 Selecting an Experiment Type

The Allele Discrimination - DNA Binding Dye Experiment Type

The Allele Discrimination - DNA Binding Dye experiment type is used to discriminate between two alleles of a gene in a genomic DNA or cDNA sample using a double- stranded DNA binding dye, such as SYBR Green or EvaGreen dye, and a high- resolution melt (HRM).

HRM analysis is a technique used for genotyping samples that include a single nucleotide polymorphism (SNP) in the DNA sequence. Applications that may use HRM analysis include species identification, mutation screening, and haplotype characterization. When using the allele discrimination experiment type for HRM analysis, you set up the experiment to amplify all alleles in the same well using the same set of primers, and the program detects all alleles using the same double- stranded DNA- binding dye (such as SYBR Green or EvaGreen dye). The thermal profile includes a melt segment so that melt curves of the targets can be generated. Even DNA amplicons that differ in sequence by only a single nucleotide will yield slightly different melt curves. An Allele Discrimination experiment that uses HRM analysis needs to include positive control samples for each base pair possibility at the SNP location (homozygous as well as heterozygous positive control samples).

On the Graphical Displays screen, Difference Plots show the difference in fluorescence between two plots during a melt ramp (Y axis) as a function of temperature (X axis). By graphing the difference in fluorescence, the program can detect even slight differences between two melt curves. You can use the difference plots to compare samples of unknown genotype to positive control samples, and visually determine which genotype group the target in the unknown sample falls into.

NOTE

A separate HRM software license is required in order to view the Difference Plots. To purchase a software license, contact your Agilent Sales representative.

About HRM calibration plates

During the HRM segment of the thermal profile, the instrument ramps the temperature of the thermal block in 0.2°C increments. At any given target temperature, very slight differences in the exact temperature may exist from well to well across the thermal block. The purpose of an HRM calibration plate (HCP) is to calculate an offset temperature for each well

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Selecting an Experiment Type 5

The Allele Discrimination - DNA Binding Dye Experiment Type

in order to help normalize these temperature variations. The offset temperature is the difference between the Tm (melting temperature) in any given well and the average Tm for the plate. When you associate your Allele Discrimination experiment with an HCP experiment, the program subtracts the offset temperature from the calculated Tm in each well. This normalization improves the accuracy and clarity of the difference plots and the raw/derivative melt curves.

In order to calculate the offset temperatures, an HCP must contain the identical amplicon in each well. During the HCP run, that amplicon is melted in an HRM segment. The program then calculates a Tm for each well and an average Tm for the plate.

All HCP experiments must be set up and run directly from the instrument (you cannot set up an HCP experiment from your PC). See “Run an HRM

calibration plate (HCP)” on page 38 for instructions.

You can use the same HCP experiment for multiple Allele Discrimination experiments. For each instrument, Agilent recommends running a new HCP experiment at least once per year.

Well types for Allele Discrimination - DNA Binding Dye experiments

Well Type Description

Unknown Contains a complete reaction mixture including a test template that

contains an unknown amount of the specific target.

NTC No template control; contains all reaction components except the

template nucleic acid.

This control is useful for detecting amplicon contamination.

Homo Allele A Contains a complete reaction mixture with a positive control template

that is known to be homozygous for allele A.

Home Allele B Contains a complete reaction mixture with a positive control template

that is known to be homozygous for allele B.

Hetero Contains a complete reaction mixture with a heterozygous positive

control template that is known to include both allele A and allele B.

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5 Selecting an Experiment Type

The Allele Discrimination - Fluorescence Probe Experiment Type

The Allele Discrimination experiment type is used to discriminate between two alleles of a gene in a genomic DNA or cDNA sample. An Allele Discrimination experiment can be performed two different ways: using fluorescent probes (as described below) or using a double- stranded DNA binding dye.

Using the probe method for allele discrimination, two fluorescent probes labeled with two spectrally distinct dyes are used to discriminate between the two alleles and, subsequently, determine the genotype of a sample. For example, if the program detects amplification in an unknown DNA sample for the dye identifying the wild- type allele but not for the dye identifying a mutant allele, the program designates the sample as wild- type homozygous. If the program detects amplification in an unknown DNA sample for the dye identifying the mutant allele but not for the dye identifying the wild- type allele, the program designates the sample as mutant homozygous. If the program detects amplification for both dyes, it designates the unknown sample as heterozygous for the two alleles. With properly designed probes, this assay is sensitive enough to detect a single- base difference (single nucleotide polymorphism or SNP) between two alleles.

The program uses the quantification cycle (Cq) value for each target in each sample to determine the genotypes of the samples. A Cq value of 24 to 32 is expected for a sample that contains the specific allele recognized by the probe. A Cq value equal to the final cycle of the PCR reaction (typically 40) or equal to the Cq of the negative control samples indicates the absence of a specific allele. The program displays the results in the Allele Determination graph, which is a scatter plot that shows the Cq for the dye specific to one allele plotted against the Cq for the dye specific to the other allele. The program groups plotted points according to their positions on the graph, providing a convenient visualization of samples which share the same genotype (allelic composition).

In the analysis of real- time Allele Discrimination experiments that use fluorescence probes (e.g., TaqMan probes), you would typically monitor and report fluorescence at the end of the annealing/extension step. At that point, the polymerase has already extended across the template and hydrolyzed any probe that had annealed.

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Selecting an Experiment Type 5

The Allele Discrimination - Fluorescence Probe Experiment Type

Well types for Allele Discrimination - Fluorescence Probe experiments

Well Type Description

Unknown Contains a complete reaction mixture including a test template that

contains an unknown amount of the specific target.

NTC No template control; contains all reaction components except the

template nucleic acid.

This control is useful for detecting amplicon contamination.

Homo Allele A Contains a complete reaction mixture with a positive control template

that is known to be homozygous for allele A.

Home Allele B Contains a complete reaction mixture with a positive control template

that is known to be homozygous for allele B.

Hetero Contains a complete reaction mixture with a heterozygous positive

control template that is known to include both allele A and allele B.

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5 Selecting an Experiment Type

The User Defined Experiment Type

The User Defined experiment type provides the greatest flexibility in setup and analysis of an experiment. All the well types and other plate setup options that are available across the other three experiment types are available on the Plate Setup screen in a User Defined experiment. Similarly, on the Thermal Profile screen, you can add any type of segment to the thermal profile, and on the Graphical Displays screen, you can view the results for any of the experiment type- specific graphs.

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6 Setting Up the Plate

Overview of the Plate Setup screen 73

The plate map 74 Well type 74 Well name / Sample name 74 Target information 75 Replicate number 75 Reference dye 75 The Properties panel 76

Additional tools for setting up a plate 76 Import a plate setup 78 Select and view wells in the plate map 79

Select wells in the plate map 79 Unselect wells in the plate map 80

View details of a well or wells 80 Export the plate map image 85 Assign plate properties for a Quantitative PCR DNA Binding Dye

experiment 86 Assign plate properties for a Quantitative PCR Fluorescence Probe

experiment 94 Assign plate properties for a Comparative Quantitation experiment 102 Assign plate properties for an Allele Discrimination DNA Binding Dye

experiment 113 Assign plate properties for an Allele Discrimination Fluorescence Probe

experiment 122 Assign plate properties for a User Defined experiment 131

Agilent Technologies

6 Setting Up the Plate

Overview of the Plate Setup screen

The Plate Setup screen has tools for assigning properties to the wells of the plate so that the program can properly analyze your data.

To open the Plate Setup screen: With an experiment or project file open, click Plate Setup in the Experiment Area panel on the left side of the screen.

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The plate map

In the center of the Plate Setup screen is a representation of the wells of a 96- well plate. This diagram, called a plate map, is used for showing which wells in the plate are in use in the current experiment, what kind of sample is in each well, which dyes are being used for detection in the wells, and what targets those dyes are detecting.

The properties assigned to the wells are indicated in the plate map. To see more detailed information on the properties of a particular well, such as target names and standard quantities, hover your cursor over the well.

Setting Up the Plate 6

Overview of the Plate Setup screen

You can also open a mini plate map to view the details in a selected set of wells.

Well type

All wells that are in- use in the experiment need to be assigned a well type. This assignment indicates to the program the type of reaction in the well. For example, the Unknown well type is used for experimental templates in which the quantity of the target(s) is unknown. When the Show setting on the Plate Setup Properties panel is set to Type, the well type appears at the top of the well.

The available well types vary depending on the experiment type.

Well name / Sample name

If desired, you can assign names to the wells of the plate. When the Show setting on the Plate Setup Properties panel is set to Name, the well name appears at the top of the well. The Comparative Quantitation, Allele

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6 Setting Up the Plate

Overview of the Plate Setup screen

Discrimination, and User Defined experiment types also allow you to assign sample names to designate the template sample used in each well. The program displays the sample name at the top of each well when the Show setting is set to Sample.

Target information

All experiment types require, at a minimum, that you designate the dyes being used for detection in the wells. You may also assign a name to the target being detected by each dye. If the same dye will be used to amplify multiple targets in the experiment, assigning a unique name to each target allows the program to distinguish between these targets during analysis.

Replicate number

If some of the wells on the plate are technical replicates of each other (i.e., they have the exact same reaction components), you can designate the replicate wells during plate setup by assigning them the same replicate number. During analysis, you can choose to have the results from replicate wells averaged together at each cycle, or you can treat replicates separately to monitor for well- to- well variation among identical reactions.

Invalid Sets: When assigning replicates, if you see a flashing red warning icon in the Properties panel next to Replicates, you have an invalid replicate set on the plate. To be valid, all the wells of a set must be of the same well type and have the same target assignments. Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid.

Note that technical replicates are different from biological replicates. The assignment of biological replicate IDs is unique to Comparative Quantitation (and User Defined) experiments.

Reference dye

Passive reference dyes are used for normalization of the fluorescence signal in order to compensate for non- PCR related variations in fluorescence, such as pipetting variation from well to well. Typically, most experiments use ROX as the reference dye.

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If you will be adding a dye to the wells of your plate as a passive reference dye, you need to assign a reference dye in the plate setup. The assignment of that dye as a reference dye is indicated in the wells by the target name REF. Note that if you assign a reference dye, you must assign it to all wells in use on the plate.

The Properties panel

The panel on the right side of the Plate Setup screen has the tools for assigning properties to the wells in the plate map. The content of this panel depends on the experiment type. See the following help topics for information on your experiment type:

“Assign plate properties for a Quantitative PCR DNA Binding Dye experiment” on page 86

“Assign plate properties for a Quantitative PCR Fluorescence Probe experiment” on page 94

“Assign plate properties for a Comparative Quantitation experiment” on

page 102

Setting Up the Plate 6

Overview of the Plate Setup screen

“Assign plate properties for an Allele Discrimination DNA Binding Dye experiment” on page 113

“Assign plate properties for an Allele Discrimination Fluorescence Probe experiment” on page 122

“Assign plate properties for a User Defined experiment” on page 131

To hide the Properties panel, click the arrow icon in the upper left corner of the panel. Click the arrow again to display the Properties panel.

Additional tools for setting up a plate

Copy/Paste

To copy well information, first select the wells that contain the information to be copied. Then, press Ctrl+c, or right- click on the plate map and click Copy, to copy the properties of the selected wells to the clipboard. You can later paste the properties into other wells within the same experiment or in another open experiment.

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6 Setting Up the Plate

Overview of the Plate Setup screen

To paste well information from the clipboard to a set of wells, first select the wells that you want to paste into. Then, press Ctrl+v, or right- click on the plate map and click Paste, to paste the well information into a selected set of wells. You can paste into wells within the same experiment, or into wells of an experiment that is open in another tab of the program (provided that the properties of the wells are compatible with the experiment type).

See “Select and view wells in the plate map” on page 79 for instructions on selecting sub- sets of wells on the plate map.

Undo/Redo

Click the Undo icon in the lower right corner of the screen to undo your most recent action. You can click the icon multiple times to undo multiple, consecutive actions.

Click the Redo icon in the lower right corner of the screen to redo the most recent action that you reversed using the Undo tool. You can click the icon multiple times to redo multiple, consecutive actions.

You can also access the Undo and Redo commands by right- clicking anywhere on the plate map.

Clear Selected Well or Clear Plate

To clear all the properties assigned to a well or set of wells, select the well(s) on the plate map, right- click, and then click Clear Selected Well. Alternatively, select the well(s) on the plate map and press Delete to clear the properties.

To clear all properties assigned to all wells in the plate, right- click anywhere on the plate map and click Clear Plate.

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Import a plate setup

The Aria program allows you to set up the plate on the Plate Setup screen by importing the plate setup of any existing experiment of the same experiment type. After you import the plate setup, you can edit the well properties as desired.

To open the Plate Setup screen: With an experiment or project file open, click Plate Setup in the Experiment Area panel on the left side of the screen.

To import a plate setup:

1 In the Properties panel of the Plate Setup screen, click the Import Plate

Setup icon.

The Open dialog box opens.

2 Browse to the folder of the existing experiment with the plate setup

that you want to import. Select the file and click Open.

The dialog box closes and the program imports the plate setup into the currently open experiment.

Setting Up the Plate 6

Import a plate setup

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6 Setting Up the Plate

Select and view wells in the plate map

When setting up your plate on the Plate Setup screen, you may need to select a sub- set of wells in the 96- well plate map in order to assign properties to specific wells. You can select and unselect wells by clicking directly on the plate map. You may also need to view certain wells of the plate map in more detail, which can also be done from the Plate Setup screen.

To open the Plate Setup screen: With an experiment or project file open, click Plate Setup in the Experiment Area panel on the left side of the screen.

Select wells in the plate map

When a new experiment is first opened, all wells on the plate are already selected. Selected wells appear highlighted on the plate map (see wells A1 through A6 in the image below) while unselected wells appear grayed out (see wells A7 through A12 in the image below).

To select all wells when some wells are not already selected:

1 Click the small square in the upper left- hand corner of the plate. (If all

wells on the plate are already selected, clicking this button will unselect all wells.)

To select an entire row or column of wells:

1 Click the corresponding row header (A–H) or column header (1–12).

To select a range of adjacent wells:

1 Click and hold the left mouse button as you drag the cursor across the

wells to be selected.

A visible marking rectangle appears.

79 Agilent Aria Real-Time PCR Program

2 When all of the required wells are included in the rectangle, release the

left mouse button.

The range of wells is selected.

To select a group of non- contiguous wells:

1 Press Ctrl as you click individually on each of the wells to be selected.

Unselect wells in the plate map

Use one of the following approaches to unselect wells:

• To unselect individual wells, press Ctrl as you click on the wells to be unselected.

• To unselect a selected row or column, click on the header for that row or column.

• To rapidly unselect all wells, click twice on the button in the upper left- hand corner of the plate. This will select and then unselect all wells.

Setting Up the Plate 6

Select and view wells in the plate map

View details of a well or wells

When the plate map is displaying all 96 wells, details such as target names and standard quantities are not displayed. The Plate Setup screen offers multiple ways to view the detailed properties of a single well or multiple wells.

View details of an individual well

To view the detailed properties of an individual well:

• Hover the cursor over the well.

The program opens a larger schematic of the well that displays the properties assigned to the well.

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6 Setting Up the Plate

Select and view wells in the plate map

Zoom in/out on the plate map

You can zoom in and zoom out on the wells of the plate map using the commands on the plate map short- cut menu.

To zoom in on the wells of the plate map:

1 Right- click anywhere on the plate map.

The short- cut menu opens.

2 Click Zoom In.

The program zooms in on the plate map, displaying fewer wells in more detail.

3 If desired, repeat steps 1- 2 to zoom in further.

To zoom out on the wells of the plate map:

1 Right- click anywhere on the plate map.

The short- cut menu opens.

2 Click Zoom Out.

The program zooms out on the plate map.

3 If desired, repeat steps 1- 2 to zoom out until the plate map displays all

96 wells.

View a mini plate map

The minimap tool allows you to limit the plate map to specific wells, which enables you to view more detailed information on the properties of those wells.

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Setting Up the Plate 6

Select and view wells in the plate map

To view a mini plate map:

1 In the Properties panel, click the Minimap icon.

The Minimap box opens. This box displays a small schematic of the plate map.

2 In the Minimap box, click the zoom scroll bar to zoom in on the wells

in the minimap.

The red box outlines the wells to be included in the minimap.

3 Click and drag the red box to capture the wells that you want to view

in more detail.

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6 Setting Up the Plate

Select and view wells in the plate map

4 Click the X in the upper right corner of the Minimap box to close the

box.

The plate map on the Plate Setup screen displays only the wells selected in the Minimap box.

To restore the mini plate map to the full 96 wells:

1 In the Properties panel, click the Minimap icon.

The Minimap box opens.

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Setting Up the Plate 6

Select and view wells in the plate map

2 Click the Maximize icon in the lower left corner of the Minimap box.

3 Click the X in the upper right corner of the Minimap box to close the

box.

The plate map displays all 96 wells.

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6 Setting Up the Plate

Export the plate map image

The image of the Plate Setup screen's plate map can be exported to a Microsoft PowerPoint presentation.

To open the Plate Setup screen: With an experiment or project file open, click Plate Setup in the Experiment Area panel on the left side of the screen.

To export an image of the plate map to PowerPoint:

• From the Plate Setup screen, right- click anywhere on the plate map. In the short- cut menu, click Send Image to PowerPoint.

PowerPoint opens to a new presentation file with the plate map image on the slide.

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Setting Up the Plate 6

Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

The controls on Plate Setup screen's Properties panel allow you to create a customized plate setup for experiments of the type Quantitative PCR - DNA Binding Dye Including Standard Melt.

To open the Plate Setup screen: When you create a new Quantitative PCR experiment, you will automatically be directed to the Plate Setup screen. To return to the Plate Setup screen at any time before, during, or after a run, click Plate Setup in the Experiment Area panel on the left side of the screen.

Assign well types

Use the Well Types drop- down list to assign well types to all the wells used in the experiment. See “Well types for Quantitative PCR experiments” on page 62 for a description of the available well types.

To assign well types:

1 On the Plate Setup screen, select all the wells in the plate map that are

of the same type. (For instructions on well selection, see “Select and

view wells in the plate map” on page 79.)

2 Select a well type from the Well Types drop- down list in the Properties

panel.

When the Show setting on the Properties panel is set to Type, the well type appears at the top of the selected wells.

3 Repeat steps 1- 2 for all well types to be included in the experiment.

Assign well names

After you assign wells to a well type you can, if desired, assign custom well names. Well names can be assigned manually or they can be imported from an Excel spreadsheet or comma-delimited text file.

Assign wells to a well type before assigning well names.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

To assign well names manually:

1 On the Properties panel of the Plate Setup screen, next to Show, select

Name.

The Well Name field becomes available for typing.

2 Select all the wells in the plate map that you want to assign to the

same well name. (For instructions on well selection, see “Select and

view wells in the plate map” on page 79.)

3 In the Well Name field, type the well name for the selected wells. Press

Enter.

The Well Name appears at the top of the selected wells.

Well names must not start with the number zero (0).

4 Repeat steps 2- 3 for all well names that you want to assign.

To assign well names by importing an Excel spreadsheet or comma- delimited text file:

1 Create the Excel spreadsheet or comma- delimited text file and save it

to a location that is accessible while working in the Aria software.

The file must be formatted as shown below with well IDs on the left and well names on the right. The well IDs may appear in any order but must use the syntax A1—H12.

2 On the Plate Setup screen, right- click on the plate map. In the menu

that opens, click Import Well Name.

The Open dialog box opens.

3 At the bottom of the dialog box, use the drop- down list to select the

appropriate file type (Text, Excel Workbook, or Excel 97- 2003 Workbook).

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Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

4 Browse to the file created in step 1. Select the file and click Open.

The software imports the well names from the file into the experiment. A message box opens notifying you that the import was successful.

5 Click OK in the message box to close it.

The plate map displays the imported well names. For any wells that have not yet been assigned a well type, the well name remains blanks until a well type is assigned.

Assign dyes/targets

Use the check boxes, drop-down lists, and fields under Assign Dyes/Targets to indicate which dyes are being used in each well and what

target each dye is detecting. Dye assignments are required, but target name assignments are optional. If different wells will be using the same

dye to detect different targets, assigning a unique name to each target enables the program to treat each target separately during analysis.

Setting Up the Plate 6

To assign dyes and target names:

1 On the Plate Setup screen, select all the wells in the plate map that

contain the same target. (For instructions on well selection, see “Select

and view wells in the plate map” on page 79.)

2 In the Properties panel, under Add Dyes, mark the Use check box for

the dye used for target detection in the selected wells.

3 If the fields for entering target names are not displayed, click the arrow

next to Targets.

The fields appear to the right of the dye names.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

4 For the marked dye, type a name into the adjacent Target Name field.

The program assigns the target name to the selected wells.

If you do not assign a target name, the program uses the dye name as the target name.

5 Repeat steps 1- 4 for all wells included in use in the experiment.

6 (Optional) Select a new color to associate with a target:

a Click the colored dot to right of the Target Name field.

b In the selection box that opens, click the desired color, or click

Advanced for more color options.

Select a reference dye

You can include a reference dye (e.g., ROX dye) to normalize the fluorescence signal of the reporter dye.

To assign a reference dye:

• On the Plate Setup screen, select the reference dye from the Reference Dye drop- down list in the Properties panel.

The program assigns the target name REF to all wells in use in the experiment and displays an R ( ) in the wells of the plate map to indicate that the well contains a reference dye.

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Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

Assign replicates

The Aria program uses replicate ID numbers to denote technical replicates. Technical replicates are QPCR reaction tubes containing identical reaction components and set up using a template from the exact same biological sample source. While biological replicates measure the variability in the experimental results due to uncontrolled biological variation from sample to sample, technical replicates are used to measure the variability in results that is introduced during the process of experimental setup.

You can assign replicates using the Manual option or the Auto option. When you designate replicate wells on the Plate Setup screen, you can set the analysis criteria to average results from those wells or treat the wells separately.

Setting Up the Plate 6

NOTE

When assigning replicates, if you see a flashing red warning icon in the Properties panel next to Replicates, you have an invalid replicate set on the plate. To be valid, all the wells of a set must be of the same well type and have the same target assignments. Hover your cursor over the warning icon to view specific information on why the program has called a replicate set invalid.

To assign replicates with the Auto option:

1 On the Plate Setup screen, select all the wells on the plate map that

have the same number of wells per replicate set.

2 In the Properties panel under Replicates, select Auto.

3 In the Direction of Assignment drop- down list, specify how the

replicate wells are arranged on the plate.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

• Select Horizontal if the replicate reactions will be arranged

horizontally in rows.

• Select Vertical if the replicate reactions will be arranged vertically in

columns.

4 In the Wells per replicate set field, type the number of replicate wells

per reaction, or click the +/- buttons to enter the desired number.

The assigned replicate number appears in each selected well.

5 If desired, make adjustment to the auto- assignments in any of the wells

of the plate by switching to the Manual option and manually assigning replicate numbers to those wells.

To manually assign replicates:

1 On the Plate Setup screen, select a set of wells that are part of the

same replicate set. Make sure that the selected wells are of the same well type and contain identical targets. (For instructions on well selection, see “Select and view wells in the plate map” on page 79.)

2 In the Properties panel under Replicates, select Manual (if not already

selected).

3 In the Assign Replicate Number field, type in the desired replicate

number for the selected wells, or click the +/- buttons to enter the desired number.

The assigned replicate number appears in each selected well.

To assign replicates using Auto Increment:

1 On the Plate Setup screen, assign well types as needed for your

experiment.

2 In the Properties panel under Replicates, select Manual (if not already

selected).

3 Click Auto Increment.

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Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

When you hover your cursor anywhere on the plate map, an icon of the number 1 appears next to the cursor.

4 With the cursor, click and drag across the group of wells that you want

to assign as replicate number 1.

The program assigns the wells to replicate number 1, and the icon next to the cursor changes to a number 2.

5 Click and drag across the group of wells that you want to assign as

replicate number 2.

The program assigns the wells to replicate number 2, and the icon next to the cursor changes to a number 3.

6 Continue assigning replicate numbers for the remainder of the plate.

When finished, click Auto Increment to turn off the Auto Increment function.

Assign quantities to Standard wells

In order to generate a standard curve from your data, you need to assign the initial template quantity to each Standard well.

Setting Up the Plate 6

To assign the standard quantities for a target in the Standard wells:

1 On the Plate Setup screen, select the Standard wells. (For instructions

on well selection, see “Select and view wells in the plate map” on page 79.)

2 In the Properties panel under Standard Quantities, select which target

in the selected wells is the standard target (i.e., the target of known quantity in the template).

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Assign plate properties for a Quantitative PCR DNA Binding Dye experiment

3 In the Starting Amount field, type in the quantity of the standard target

present in the first replicate set of Standard wells. You will be asked to specify the units for this amount in step 5.

NOTE

This quantity must be either the highest quantity or the lowest quantity in the dilution series of the standard template sample.

4 In the drop- down list labeled A factor of, select the dilution factor

used to generate the dilution series of the standard template. For example, if each standard quantity is separated by a factor of 10, select 10x. Negative dilution factors are used to specify a decrease in quantity from the starting amount while positive dilution factors specify an increase from the starting amount.

5 In the drop- down list labeled Units (for Plate), select the units of the

quantity entered in the Starting Amount field. Note that all Standard wells on the plate must use the same units.

To clear the assigned standard quantity from one or more wells:

• Select the well(s) and click Clear.

93 Agilent Aria Real-Time PCR Program

Setting Up the Plate 6

Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

The controls on Plate Setup screen's Properties panel allow you to create a customized plate setup for experiments of the type Quantitative PCR - Fluorescence Probe.

Assign well types

To assign well types:

1 On the Plate Setup screen, select all the wells in the plate map that are

of the same type. (For instructions on well selection, see “Select and

view wells in the plate map” on page 79.)

2 Select a well type from the Well Types drop- down list in the Properties

panel.

When the Show setting on the Properties panel is set to Type, the well type appears at the top of the selected wells.

3 Repeat steps 1 and 2 for all well types to be included in the

experiment.

Assign well names

Assign wells to a well type before assigning well names.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

To assign well names manually:

1 On the Properties panel of the Plate Setup screen, next to Show, select

Name.

The Well Name field becomes available for typing.

2 Select all the wells in the plate map that you want to assign to the

same well name. (For instructions on well selection, see “Select and

view wells in the plate map” on page 79.)

You must assign a well to a well type before you can assign it a well name.

3 In the Well Name field, type the well name for the selected wells. Press

Enter.

The Well Name appears at the top of the selected wells.

Well names must not start with the number zero (0).

4 Repeat steps 2- 3 for all well names that you want to assign.

To assign well names by importing an Excel spreadsheet or comma- delimited text file:

1 Create the Excel spreadsheet or comma- delimited text file and save it

to a location that is accessible while working in the Aria software.

The file must be formatted as shown below with well IDs on the left and well names on the right. The well IDs may appear in any order but must use the syntax A1—H12.

2 On the Plate Setup screen, right- click on the plate map. In the menu

that opens, click Import Well Name.

The Open dialog box opens.

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Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

3 At the bottom of the dialog box, use the drop- down list to select the

appropriate file type (Text, Excel Workbook, or Excel 97- 2003 Workbook).

4 Browse to the file created in step 1. Select the file and click Open.

The software imports the well names from the file into the experiment. A message box opens notifying you that the import was successful.

5 Click OK in the message box to close it.

The plate map displays the imported well names. For any wells that have not yet been assigned a well type, the well name remains blanks until a well type is assigned.

Assign dyes/targets

Use the check boxes, drop-down lists, and fields under Assign Dyes/Targets to indicate which dyes are being used in each well and what

target each dye is detecting. Dye assignments are required, but target name assignments are optional. If different wells will be using the same

dye to detect different targets, assigning a unique name to each target enables the program to treat each target separately during analysis.

Setting Up the Plate 6

To assign dyes and target names:

1 On the Plate Setup screen, select all the wells in the plate map that

contain the same target. (For instructions on well selection, see “Select

and view wells in the plate map” on page 79.)

2 Under Add Dyes, mark the Use check boxes for the dyes used for target

detection in the selected wells.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

3 If the fields for entering target names are not displayed, click the arrow

next to Targets.

The fields appear to the right of the dye names.

4 For the marked dyes, type a name into the adjacent Target Name field.

The program assigns the target names to the selected wells.

If you do not assign a target name to a marked dye, the program uses the dye name as the target name.

5 Repeat steps 1- 4 for all wells included in use in the experiment.

6 (Optional) Select a new color to associate with a target:

a Click the colored dot to right of the Target Name field.

b In the selection box that opens, click the desired color, or click

Advanced for more color options.

Select a reference dye

You can include a reference dye (e.g., ROX dye) to normalize the fluorescence signal of the reporter dye.

To assign a reference dye:

• On the Plate Setup screen, select the reference dye from the Reference Dye drop- down list in the Properties panel.

The program assigns the target name REF to all wells in use in the experiment and displays an R ( ) in the wells of the plate map to indicate that the well contains a reference dye.

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Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

Assign replicates

Replicates are wells that contain identical reaction components (repeats). You can assign replicates using the Manual option or the Auto option. When you designate replicate wells on the Plate Setup screen, you can set the analysis criteria to average results from those wells or treat the wells separately.

Setting Up the Plate 6

NOTE

To assign replicates with the Auto option:

1 On the Plate Setup screen, select all the wells on the plate map that

have the same number of wells per replicate set.

2 In the Properties panel under Replicates, select Auto.

3 In the Direction of Assignment drop- down list, specify how the

replicate wells are arranged on the plate.

• Select Horizontal if the replicate reactions will be arranged

horizontally in rows.

• Select Vertical if the replicate reactions will be arranged vertically in

columns.

4 In the Wells per replicate set field, type the number of replicate wells

per reaction, or click the +/- buttons to enter the desired number.

The assigned replicate number appears in each selected well.

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6 Setting Up the Plate

Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

5 If desired, make adjustment to the auto- assignments in any of the wells

of the plate by switching to the Manual option and manually assigning replicate numbers to those wells.

To manually assign replicates:

1 On the Plate Setup screen, select a set of wells that are part of the

2 In the Properties panel under Replicates, select Manual (if not already

selected).

3 In the Assign Replicate Number field, type in the desired replicate

number for the selected wells, or click the +/- buttons to enter the desired number.

The assigned replicate number appears in each selected well.

To assign replicates using Auto Increment:

1 On the Plate Setup screen, assign well types as needed for your

experiment.

2 In the Properties panel under Replicates, select Manual (if not already

selected).

3 Click Auto Increment.

When you hover your cursor anywhere on the plate map, an icon of the number 1 appears next to the cursor.

4 With the cursor, click and drag across the group of wells that you want

to assign as replicate number 1.

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Assign plate properties for a Quantitative PCR Fluorescence Probe experiment

The program assigns the wells to replicate number 1, and the icon next to the cursor changes to a number 2.

5 Click and drag across the group of wells that you want to assign as

replicate number 2.

The program assigns the wells to replicate number 2, and the icon next to the cursor changes to a number 3.

6 Continue assigning replicate numbers for the remainder of the plate.

When finished, click Auto Increment to turn off the Auto Increment function.

Assign quantities to Standard wells

In order to generate a standard curve from your data, you need to assign the initial template quantity to each Standard well.

Setting Up the Plate 6

To assign the standard quantities for a target in the Standard wells:

1 On the Plate Setup screen, select the Standard wells. (For instructions

on well selection, see “Select and view wells in the plate map” on page 79.)

2 In the Properties panel under Standard Quantities, select which target

in the selected wells is the standard target (i.e., the target of known quantity in the template).

3 In the Starting Amount field, type in the quantity of the standard target

present in the first replicate set of Standard wells. You will be asked to specify the units for this amount in step 5.

NOTE

Agilent Aria Real-Time PCR Program 100

This quantity must be either the highest quantity or the lowest quantity in the dilution series of the standard template sample.

Agilent Aria Real-Time PCR User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

The Aria software 15 Getting Started with the Aria Software 16

The Aria software

Getting Started with the Aria Software

Introduction to the Aria software

Overview of the user interface

Home screen

Menu toolbar

Tabs

Left and right panels

Home/Notifications/Help icons

Getting Started screen

Experiment Notes / Project Notes

Help access for the Aria software

Help system

Sample experiments

Update instrument optics

Change the default crosstalk correction settings

Set software preferences

Set default file name configuration

Create and apply analysis templates

Performing Hardware and Software Tests and HRM Calibrations

Run an instrument qualification test

Run the test

About the Qualification Test graphical data

Perform an Installation Qualification test

Run an HRM calibration plate (HCP)

Prepare the plate

Run an HCP

If the HCP fails

Overview of the Getting Started screen

Tools available on the Getting Started screen

About the Aria file types

Quick Start Protocol

How to create, set up, run, analyze, and generate reports for an experiment

Create a new experiment

Create an experiment based on experiment type

Create an experiment from a template

Create an experiment from a LIMS data file

Open an existing experiment

Save a copy of an existing experiment

Create a template from an existing experiment

Convert an experiment to a new experiment type

Selecting an Experiment Type

Overview of Experiment Types

Quantitative PCR

Comparative Quantitation

Allele Discrimination - Fluorescence Probes

Allele Discrimination - DNA Binding Dye with High-Resolution Melt

User Defined

The Quantitative PCR Experiment Type

Multiplexing quantitative PCR experiments

Well types for Quantitative PCR experiments

The Comparative Quantitation Experiment Type

Normalizing chance variations in target levels

Determining amplification efficiencies for the targets of interest and normalizer targets

Including biological replicates in comparative quantitation

Well types for Comparative Quantitation experiments

The Allele Discrimination - DNA Binding Dye Experiment Type

About HRM calibration plates

Well types for Allele Discrimination - DNA Binding Dye experiments

The Allele Discrimination - Fluorescence Probe Experiment Type

Well types for Allele Discrimination - Fluorescence Probe experiments

The User Defined Experiment Type

Overview of the Plate Setup screen

The plate map

Well type

Well name / Sample name

Target information

Replicate number

Reference dye

The Properties panel

Additional tools for setting up a plate

Import a plate setup

Select and view wells in the plate map

Select wells in the plate map

Unselect wells in the plate map