Agilent’s MX QPCR Software Tutorial

Cathy CutlerField Application Scientist

Agilent-Genomics

Our measure is your success.

General Functionality of MX Pro Software

MxPro 4.1General 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 OrganizationUser-friendly navigation

MxPro 4.1General 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 4.1General 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 4.1General functionality

Default Sets options:

Apply to new experiments

Apply to opened experiments

MxPro 3.x preferencesMxPro 4.1 preferences

MxPro 4.1General 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 plotsMxPro 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

Standalone Instrument-Connected

Goal: Easy dropdown provides access to dye list without having to go to Optics Configuration menu.

Right Mouse-Click Functionality

Ease-of-Use: Added 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

Normalizer

Comparative Quantification in Mx SoftwareGiven two samples one gene and one normalizer: What is the difference in gene expression?

Control (Calibrator) Sample 1 to x#

Gene of Interest CtCt

CtCt

GOI Ratio

Correction Factor

Normalized sample GOI fold change compared to control

Normalizer

Comparative Quantification in MxPro SoftwareGiven 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 CtCt

CtCt

GOI Ratio for Gene A

Single Correction Factor

Normalized sample GOI fold change for Gene A, B, and C compared to control

Gene of Interest: B CtCt

Gene of Interest: C CtCt

GOI Ratio for Gene B

GOI Ratio for Gene C

Comparative QuantitationPlate 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.

Treating technical replicates separately

Comparative QuantitationRelative Quantity Charts

Treating technical replicates collectivelyAdd error bars to relative quantity chart

View fold change chartExport chart to Excel

Comparative QuantitationRelative Quantity Charts

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 FormatsText Report

Excel spreadsheetPowerPoint presentation

Any screen shot

Generate Excel Charts when Exporting Charts

Provide modifiable Excel charts for biologist customization and ease of use.

Dissociation Curve

-17

3

23

43

63

83

103

123

143

163

183

55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95

Temperature (°C)

Flu

ore

scen

ce (

-R' (

T))

Dual Color Scatter Plot

21.43

22.43

23.43

24.43

25.43

26.43

27.43

28.43

29.43

30.43

31.43

32.43

33.43

34.43

35.43

36.43

25.36 26.36 27.36 28.36 29.36 30.36 31.36 32.36 33.36 34.36 35.36 36.36

Ct (using dRn) for TET

Ct

(usi

ng

dR

n)

for

FA

M

Standard Curve

19

20

21

22

23

24

100 1000 10000 100000

Initial Quantity (copies)

Ct

(dR

n)

SYBR Standards, RSq:0.998

SYBR Unknowns

SYBR, Y = -3.339*LOG(X) +33.64, Eff. = 99.3%

SYBR Unknowns

Relative Quantity Chart

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

9 10 11 12 13

Replicate/Well ID

Rel

ativ

e Q

uan

tity

(d

Rn

)

Export Amp Plots – PowerPoint or Excel

Export Std Curves – PowerPoint or Excel

Live Software Demo