Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 56)

Section 6 Data Analysis Module
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Section 6. Data Analysis Module
This section contains information on the following topics:
o
PCR Quant tab (page 48)
o
Amplification chart (page 49)
o
Standard Curve chart (page 53)
o
Results section (page 55)
o
Melt Curve and Peak charts (page 65)
o
Allelic Discrimination module (page 69)
o
End Point analysis tab (page 75)
o
Gene Expression analysis tab (page 78)
o
Gene Study: Multi file Gene Expression Analysis (page 99)
o
Post-Run Plate Editing (page 105)
o
Reports (page 107)
The Data Analysis module is where data is presented and analyzed. When the iQ5 software
opens, the Data Analysis module is gray out and inactive. To analyze a data file, open the file in
the Data File tab of the Workshop module by selecting the file and then clicking Analyze.
The Data Analysis module consists of six tabs:
•
PCR Quant: The PCR Quant tab is used to set the analysis conditions for the data file.
The analysis conditions include setting the PCR baseline and threshold, and the wells to
be excluded or included in the experiment. The analysis conditions should be set before
using the Gene Expression, End Point, or Allelic Discrimination tabs. For experiments with
standards of known quantities, the PCR Quant tab is also where absolute quantities can
be determined. The efficiency of the PCR reaction can also be determined using standard
curves with either known quantities used to produce the standard curve or by using a
serial dilution of the template under investigation
•
Melt Curve/Peak: Melt Curve/Peak is a dynamic tool used to measure the melting
temperature (T
m
) of double-stranded DNA molecules. DNA duplexes can be visualized by
either incorporation of DNA-binding dyes (for example, SYBR ® Green I) or by
hybridization with fluorescently labeled probes. Three major applications for Melt
Curve/Peak are peak identification (number of amplified products), characterization of
molecular beacons, and allelic discrimination
•
End Point : The End Point tab provides a convenient method of analyzing final RFU
(relative fluorescence unit) values. This can be useful when PCR Analysis is to determine
if a given sample is positive or negative for a particular nucleic acid sequence
•
Allelic Disc: The Allelic Disc tab is useful for assigning genotypes to unknown samples
by making comparisons to known genotypes. It can be used to distinguish among
homozygous wild types, homozygous mutants, and heterozygous individuals based either
on threshold cycle or on RFU. Allelic Discrimination analysis can only be performed on
datasets that contain multiplexed PCR data