CFX96 and CFX384 Systems Manual
115
A
: This is where the CFX Manager software calculations differ from those outlined on the
geNorm web site. In the example on that web site, the results are not scaled to the control until
normalized expression is calculated. This is referred to as re-scaled normalized expression in
the spreadsheet.
Q
: How does normalized expression, as calculated by CFX Manager software, compare to the
model introduced by M. Pfaffl (2001)?
A
: If you only evaluate one reference gene and one gene of interest, you will get exactly the
same results using the CFX Manager software as you would using the model introduced in M.
Pfaffl (2001). However, standard deviations might be slightly different.
Q
: How does normalized expression as calculated by this software compare to the model
outlined by Dr. Jo Vandesompele on the geNorm web site?
A
: The CFX Manager software uses the models outlined on the geNorm web site and will give
you the same results.
Q
: Why would I have to assign Target Names (genes) in the Gene Expression tab?
A: If you have not assigned Target Names in your initial plate setup or if you are studying more
than five genes, click the
View/Edit Plate
button to open the Plate Editor and assign target
names to the wells in the plate.
Q
: Can I customize my target (gene) and sample (condition) names?
A
: Yes. Open the Experiment Settings window (page 48) to add names to the Targets or
Samples tabs, where you can also enter or remove the full names from the lists. Alternatively,
permanently add long lists of names to the Libraries for target and sample names in the Plate
tab in the User Preferences window (page 125). These names appear on the axis in various
chart views, including gene expression.
Q
: How do I determine efficiencies?
A
: Typically the efficiency for each primer (or primer/probe) set is evaluated and recorded
during assay development. Generate a standard curve using serial dilutions of a representative
sample across a relevant dynamic range, and then record the efficiency for subsequent gene
expression analysis.
Gene Expression Calculations
CFX Manager software calculates formulas automatically and displays the resulting
information in the Data Analysis tabs.
Reaction Efficiency
Evidence suggests that using accurate measure of efficiencies for each primer and probe sets
will give you more accurate results when analyzing gene expression data. The default value of
efficiency used in the gene expression calculations is 100%. To evaluate the reaction
efficiency, generate a standard curve using serial dilutions of a representative sample across a
relevant dynamic range, and then record the efficiency for subsequent gene expression
analysis. If your experiment includes a standard curve, then the software automatically
calculates the efficiency and displays it under the Standard Curve on the Quantitation tab
when Auto Efficiency is checked in the Targets tab in the Experiment Settings window.
Summary of Contents for CFX384
Page 9: ...Table of Contents viii ...
Page 49: ...Protocols 40 ...
Page 89: ...Data Analysis Overview 80 ...
Page 149: ...Resources 140 ...
Page 157: ...Index 148 ...
