Transcript QPCR: MX Software Training- How to Set Up an Absolute

Agilent’s MX QPCR Software
Tutorial
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
MxPro3.x:
4.1:Amplification
Amplificationplots
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)
Gene of Interest
Normalizer
Ct
Ct
Sample 1 to x#
Ct
Ct
GOI Ratio
Correction
Factor
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)
Gene of Interest: A
Ct
Gene of Interest: B
GOI Ratio for
Gene A
Ct
Ct
Gene of Interest: C
Normalizer
Sample 1 to x#
Ct
Ct
Ct
GOI Ratio for
Gene B
Ct
Ct
GOI Ratio for
Gene C
Single Correction
Factor
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
PowerPoint presentation
Excel spreadsheet
Generate Excel Charts when Exporting Charts
Provide modifiable Excel charts for biologist customization and ease of use.
Dissociation Curve
183
163
143
Fluorescence (-R' (T))
123
103
Dual Color Scatter Plot
83
63
36.43
35.43
43
34.43
23
-17
55
57
59
61
63
65
67
69
71
73
75
77
79
81
83
85
87
89
91
93
Temperature (°C)
Standard Curve
Ct (using dRn) for FAM
33.43
3
32.43
31.43
30.43
95
29.43
28.43
27.43
26.43
25.43
24.43
23.43
24
22.43
21.43
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
23
Relative Quantity Chart
SYBR Standards, RSq:0.998
Ct (dRn)
22
20
19
100
1.8
SYBR, Y = -3.339*LOG(X) +
33.64, Eff. = 99.3%
1.6
SYBR Unknowns
1.4
Relative Quantity (dRn)
21
SYBR Unknowns
1000
10000
100000
1.2
1
0.8
0.6
0.4
Initial Quantity (copies)
0.2
0
9
10
11
Replicate/Well ID
12
13
Export Amp Plots – PowerPoint or Excel
Export Std Curves – PowerPoint or Excel
Live Software Demo