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

2. General Functionality of MX Pro Software

3. 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

4. MxPro Software Organization User-friendly navigation

5. 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

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

7. 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 or 3-step cycling protocol, dissociation curve
 Estimated run time shown

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

9. 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

10. 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

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

12. 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

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

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

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

16. Comparative Quantification using MX Pro Software

17. 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

18. 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

19. 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

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

21. 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.

22. 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

23. 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.

24. Comparative Quantitation Relative Quantity Charts
Treating technical replicates separately

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

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

27. 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.

28. 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.

29. 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.

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

31. Text Report Functionality in MX Pro Software

32. 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

33. 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.

34. 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.

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

36. 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.

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

38. Export Amp Plots – PowerPoint or Excel

39. Export Std Curves – PowerPoint or Excel

40. Live Software Demo