Chapter 1 Get Started
About the Example Experiments
15
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
About the
Comparative C
T
(∆∆C
T
) Example
Experiment
The objective of the comparative C
T
example experiment is to compare the expression of
EGR3, MAOB, OGDH, OSGEP, and SERPING1 in brain, liver, kidney, and universal tissues.
In the comparative C
T
example experiment:
• The samples are cDNA prepared from total RNA isolated from brain, liver, kidney,
and universal tissues.
• The targets are
EGR3, MAOB, OGDH, OSGEP, and SERPING1
.
• The reference sample is brain.
• The endogenous control is human 18S.
• The experiment is designed for singleplex PCR, where the targets (
EGR3, MAOB,
OGDH, OSGEP, and SERPING1
) and endogenous control (18S) assays are
performed in separate wells.
• Reactions are set up for 2-step RT-PCR. The High-Capacity cDNA Reverse
Transcription Kit is used for reverse transcription; the TaqMan
®
Universal PCR
Master Mix is used for PCR.
• Primer/probe sets are selected from the Applied Biosystems TaqMan
®
Gene
Expression Assays product line:
– For the target assays:
• EGR3 Assay Mix: Hs00231780_m1 (RefSeq NM_004430.2)
• MAOB Assay Mix: Hs00168533_m1 (RefSeq NM_000898.3)
• OGDH Assay Mix: Mm00803121_m1 (RefSeq NM_010956.3)
• OSGEP Assay Mix: Hs00215099_m1 (RefSeq NM_017807.2)
• SERPING1 Assay Mix: Hs00163781_m1 (RefSeq NM_001032295.1/
NM_000062.2)
– For the endogenous control assay (18S), the
18S Assay Mix: Hs99999901_s1
.
Reaction Plate Layout
The 7500 software displays the 96-well plate layout as shown below.
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