Upload
isabell-edgeworth
View
216
Download
2
Embed Size (px)
Citation preview
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