Agilent’s MX QPCR Software Tutorial Field Application Scientist Cathy Cutler Field Application Scientist Agilent-Genomics Our measure is your success.

General Functionality of MX Pro Software

MxPro 4.1 General functionality New wizard with added experiment types: EvaGreen™ (with Dissociation curve) Start project for multiple experiment analysis MxPro 3.x wizard MxPro 4.1 wizard

MxPro Software Organization User-friendly navigation

MxPro 4.1 General functionality Plate Setup: No well names in standard view in previous versions. Only in fullscreen plate view Show Well Names button

Setting Well Names Well names are accessed under the Setup Screen for each experiment, just as this would be set in a single experiment.

MxPro 4.1 General functionality Thermo Profile Setup: Custom setup as in old Mx software Predefined one click setup using Standard Design: Add RT step, normal or fast 2 or 3-step cycling protocol, dissociation curve  Estimated run time shown

MxPro 4.1 General functionality Default Sets options: Apply to new experiments Apply to opened experiments MxPro 3.x preferences MxPro 4.1 preferences

MxPro 4.1 General functionality Analysis section: Lock thresholds: Threshold is fixed to current value. Selection of different part of plate doesn‘t change threshold when locked. Customizable Toolbar Label graph according to replicate/well ID or well name MxPro 3.x: Amplification plots MxPro 4.1: Amplification plots

Improved Plate Setup functionality: Assay name/gene target identification so one dye can have multiple assay/gene names New Excel template for plate setup import Well names and quantities automatically across all replicate wells from a single entry Dye editing in plate setup Add right mouse-click capability for easier access to well names, analysis options, legend, and other related functions

Plate Setup – Importing, Well Name/ID, Assay Names

Add Dye Editing from Plate Setup Goal: Easy dropdown provides access to dye list without having to go to Optics Configuration menu. Standalone Instrument-Connected

Ease-of-Use: Added Functionality Right Mouse-Click Functionality

Right Mouse Click Functionality Plate Setup Thermal Profile Setup Raw Data Plots

Right Mouse Click Functionality Analysis Selection/Setup Charts Analysis Plates Text Report

Comparative Quantification using MX Pro Software

Enhancements in MxPro Software Improved Comparative Quantification functionality: Comparative Quantification analysis for multiple assays/genes simultaneously Addition of Relative Quantity Chart for log fold change which is the most common type of published data chart for this type of data Add error bars based on replicate variability to Relative Quantity Chart View Amplification Plots and Standard Curve on same screen

Comparative Quantification in Mx Software Given two samples one gene and one normalizer: What is the difference in gene expression? Control (Calibrator) Sample 1 to x# Gene of Interest Ct GOI Ratio Normalizer Correction Factor Ct Normalized sample GOI fold change compared to control

Comparative Quantification in MxPro Software Given two samples, multiple genes, and a single normalizer: What is the difference in gene expression? Control (Calibrator) Sample 1 to x# Gene of Interest: A Ct GOI Ratio for Gene A Gene of Interest: B Ct GOI Ratio for Gene B Gene of Interest: C Ct GOI Ratio for Gene C Normalizer Single Correction Factor Ct Normalized sample GOI fold change for Gene A, B, and C compared to control

Comparative Quantitation Plate Setup Assign assay names for analysis of multiple genes to a single normalizer (or unique normalizers) simultaneously.

Setting Assay Names Assignment of Assay Names is accessed under the Setup Screen for each experiment, just as this would be set in single experiment mode.

Comparative Quantification In a normal single plate analysis, the software associates normalizer wells to the corresponding GOI wells from the same sample using Association Symbols. In a Project these are associated together differently across experiments, instead using Well Names. Plate 1 Plate 2

Creating Associations Assignment of Associations for comparative quantification is accessed under the Setup Screen for each experiment, just as this would be set in single experiment mode.

Comparative Quantitation Relative Quantity Charts Treating technical replicates separately

Comparative Quantitation Relative Quantity Charts Add error bars to relative quantity chart Treating technical replicates collectively View fold change chart Export chart to Excel

Comparative Quantitation: Multiple Gene Targets Fold gene expression changes for six genes across two treatments with replicate error represented.

Absolute Quantification If you are comparing unknowns on one plate to a Standard curve run on a separate plate, the software will calculate quantities for your Unknowns based on that curve as long as both sets of wells have the same assay name.

Absolute Quantification of Unknowns Assignment of Assay names for absolute quantification must be the same and is accessed under the Setup Screen for each experiment.

Show Amp Plots and Standard Curve on One Screen View placement of threshold in Amplification Plots as well as the impact to Standard Curve simultaneously.

Customize Plot/Legend: Add Amp Plot Color Coding Based on Dye Show groups by dye/assay color or by well name

Text Report Functionality in MX Pro Software

Enhancements in MxPro Software Improved Text Report functionality: Addition of new columns Analysis properties displayed at top of text report Addition of text report sorting by different columns Generate customized consolidated data reports Auto generate Excel charts when exporting data

Text Report: Sort Text Report Columns Based on Plate Column Group data for reporting based on potential biologist plate setup and further customize data reports from software.

Text Report: Place Dye Analysis Parameters at Top of Text Report Reduce redundancy in text report (dye/assay specific information does not need to appear in every row of main report) and generate a cleaner report format.

Consolidated Data Reports Provide one screen data viewing and printing of all desired screens/reports.

Export Results in Multiple Formats Text Report Any screen shot Excel spreadsheet PowerPoint presentation All of the data generated as well as the results of the analysis can be exported in multiple formats. Numerical results can be exported as text files or directly opened in Microsoft Excel. All plots and graphs can also be exported into Excel or directly into PowerPoint where a new presentation can be generated with a single click of the mouse.

Generate Excel Charts when Exporting Charts Provide modifiable Excel charts for biologist customization and ease of use.

Export Amp Plots – PowerPoint or Excel

Export Std Curves – PowerPoint or Excel

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