Read iQ5 Data Analysis text version

iQ5 Software Training-Data analysis

Gene Expression Division www.bio-rad.com/amplification/

iQ5 Overview

Gene Expression Division www.bio-rad.com/amplification/

Ct: Automatic Determination

Gene Expression Division www.bio-rad.com/amplification/

3 Analysis Mode for Troubleshooting

Gene Expression Division www.bio-rad.com/amplification/

Detailed Results

Gene Expression Division www.bio-rad.com/amplification/

Melting Curve Analysis

Gene Expression Division www.bio-rad.com/amplification/

Absolute Quantization

Auto-Calculation Set up the Sample Type and Concs: Standard or Unknown

Determine Ct Value

Gene Expression Division www.bio-rad.com/amplification/

AQ Results

Gene Expression Division www.bio-rad.com/amplification/

Relative Quantization (Ct method)

Before Target gene Ct SB Ct B Reference gene Ct CB Ct CA Ct SA

After

Ct A

Ct Ratio(1+E) - 2

Ct (Ct A Ct B)

Gene Expression Division www.bio-rad.com/amplification/

Normalized Expression

· T (Livak) C

­ Assume 100% efficiency ­ Only one Ref Gene

Simple

· Pfaffl Modification

­ Accounts for efficiency differences ­ Only one Ref Gene

· Vandesompele

­ Accounts for efficiency differences ­ Allows multiple reference genes for normalization

Complex

Gene Expression Division www.bio-rad.com/amplification/

Gene Expression Analysis Ct Determination

Gene Expression Division www.bio-rad.com/amplification/

Gene Expression Tab

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Grouping

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Select Ref. Gene and Control

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Gene Expression Division www.bio-rad.com/amplification/

RQ Resluts

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Multifile Gene Expression - MFGE

· Accomplished through new file type called "Gene Study" · Created as .gxd file · .gxd files maintain Sample ID and CT information only · Over 50 data files can be imported into a Gene Study

­ Over 5,000 wells of data can be analyzed in a Gene Study using the iQ5 Gene Expression module ­ This is approx. 50 full plates (data files) of single color real-time PCR data or 25 plates of dual-color data, etc...

Gene Expression Division www.bio-rad.com/amplification/

SNP detection by Probe

Kostrikis et al. Science vol. 279 p. 1228-1229

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Allelic Discrimination

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Detailed Reports

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General QPCR Working Process -- Data analysis in iQ5

· Amplification plot

­ Reproducibility? So you need duplication or triplication.... ­ Determination Ct Value? Threshold

·

Absolute Q., Relative Q. or SNP

­ Ct or Ct ­ Allelic Discrimination

·

PCR efficiency from std. Curve

­ <100% ­ >100% ­ Dynamic range

· ·

Reproducibility

­ Duplication or triplication

Melting curve analysis

­ Primer dimmer ­ Non specific production

Gene Expression Division www.bio-rad.com/amplification/

Gene Expression Division www.bio-rad.com/amplification/

· General introduction · Instrument Operation · Data analysis

­ ­ ­ ­ Ct Melting Curve Absolute Quantification Relative Quantification

Gene Expression Division www.bio-rad.com/amplification/

iQ5 Software Data analysis

· General: Ct determination and melting curve · AQ · RQ

Gene Expression Division www.bio-rad.com/amplification/

Ct: Automatic Determination

Gene Expression Division www.bio-rad.com/amplification/

Gene Expression Division www.bio-rad.com/amplification/

Information

iQ5 Data Analysis

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