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Notes About Micro Fluidic Card Experiments
About the
Comparative C
Method of
Relative
Quantification
Significance of
the Calibrator
Sample
Design
Guidelines
12
June 23, 2003 9:39 am, MFC_QSG_text.fm
Relative gene expression values can be obtained from
7900HT system data using the Comparative C
T
quantification. In the Comparative C
relative to a calibrator sample that is used as the basis for comparative
results. Therefore, the calibrator is the 1✕ sample and all other
quantities are expressed as an n-fold difference relative to the
calibrator.
For more information on the Comparative C
Note:
relative quantification, refer to Livak & Schmittgen, 2001 and the
®
ABI P
7700 Sequence Detection System User Bulletin #2:
RISM
Relative Quantitation of Gene Expression (PN 4303859).
All Micro Fluidic Card relative quantification experiments require
data from a calibrator sample. During analysis, the ABI P
7900HT Sequence Detection System software (SDS 2.1 software)
calculates the levels of target gene expression in samples relative to
the level of expression in the calibrator. Thus, the calibrator sample is
an integral part of the relative quantification calculation because it
serves as the basis for the comparative results.
Examples of possible calibrator samples include:
• A zero timepoint sample in a time-course experiment
• An untreated sample (versus treated samples)
• A resting sample (versus activated samples)
The most important factor to consider when designing Micro Fluidic
Card experiments is how you intend to analyze your data:
• If you want to analyze your data using Applied Biosystems RQ
Manager Software, set up relative quantification plate
documents. (This is the recommended method.)
• If you want to analyze your data using a custom application, set
up absolute quantification plate documents.
IMPORTANT!
document, you cannot convert it into an absolute quantification
document. Similarly, you cannot convert an absolute quantification
document into a relative quantification document. (For more
information, see Chapter 6 of the user guide for the 7900HT system.)
Applied Biosystems Micro Fluidic Card Getting Started Guide
DRAFT
Once you have set up a relative quantification
Method for relative
T
Method, quantity is expressed
T
Method for
T
®
RISM
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