Applied Biosystems 7900HT User Manual

DRAFT
September 1, 2004 11:39 am, CH_Real-Time.fm
Chapter 6 Analyzing Real-Time Data
6-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Essential Experimental Components
Endogenous
Control
All relative quantification experiments require data from a second probe and primer
set that amplifies an endogenous control sequence. Endogenous control targets are
typically constitutive RNA or DNA sequences that are present at a statistically
consistent level in all experimental samples. By using the endogenous control as an
active reference, the data from the amplification of a messenger RNA (mRNA) target
can be normalized for differences in the amount of total RNA added to each reaction.
Examples of common endogenous controls are: GAPDH, 18S rRNA, and β-Actin.
The endogenous control can be amplified independently of the target sequences in
separate wells on the reaction plate (as singleplex or non-multiplex reactions), or in
the same well (as multiplex reactions).
IMPORTANT! For non-multiplex experiments, the reactions amplifying the
endogenous control must be located on the same plate as the target assays.
IMPORTANT! To generate a standard deviation for the relative quantity value of a
target, each plate must contain usable data from at least two replicates of the target
and endogenous control.
Calibrator
Sample
All relative quantification experiments require data from a calibrator sample. During
analysis, the SDS software calculates gene expression levels in samples relative to
the level of expression in a calibrator sample. Thus, the calibrator plays an integral
part in the calculation because it is used as the basis for the comparative results.
Examples of possible calibrator samples include:
• A zero time-point sample in a time-course experiment
• An untreated sample (versus treated samples)
• A resting sample (versus activated samples)
Note: The SDS software combines the data from replicate calibrator wells at the ∆C
T
level of the relative quantification calculation (whether the replicates are present on a
single or multiple plates).
Replicate Wells
For relative quantification studies, Applied Biosystems recommends the use of three
or more replicate reactions per sample to ensure statistical confidence.
Replicates allow you to:
• Preserve Data – If an amplification fails in one well, replicate wells can
potentially provide data. This point is especially true in the case of endogenous
controls which, upon failure, may invalidate the results for the entire plate.
• Remove Outliers – The use of replicate populations provide the opportunity for
the visualization and removal of outliers.
• Ensure Statistical Reproducibility – In general, the use of replicates ensures a
greater degree of experimental reproducibility by providing a means to identify
and refute anomalous data caused by experimental error (such as contamination,
pipetting errors, and so on).

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English