Applied Biosystems 7900HT User Manual page 125

Internal Structure
Detectors
Genomic DNA
Contamination
Single-Plex
Reactions
Quality Control
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Figure 4-6 illustrates the Low Density Array internal structure.
Fill port
Figure 4-6 TaqMan Low Density Array, Internal Structure
The Low Density Array permits the amplification of target and an external
endogenous control cDNA using fluorogenic 5´ nuclease assays. The assays consist
of two reactions, each a complete PCR system with corresponding probe and
primers.
The fluorogenic probes of the assays function as follows:
• Probes labeled with the FAM
cDNA targets.
• Probes labeled with the FAM
endogenous control.
TaqMan probes and primers for the cDNA targets span exon-exon junctions to
minimize the contribution of contaminating genomic DNA. Performance tests
demonstrate that 5´ exonuclease assays can be run with samples containing up to
10,000 copies of genomic DNA without detection of contaminants, unless otherwise
specified by Applied Biosystems.
External Endogenous Control Assay
Note that the commonly-used external endogenous control assay is not RNA-specific
and consequently is affected by genomic DNA contamination. However, because of
the extremely high expression level of rRNA, even gross DNA contamination has a
negligible effect on the relative quantification values obtained from the Low Density
Array.
The Low Density Array is recommended for use with single-plex TaqMan reagents
(for example, FAM dyes). An external endogenous control assay (for example,
FAM-18S or FAM-GAPDH) is used as a calibrator in relative quantification
calculations.
For more information on relative quantification, see ABI P
Note:
Sequence Detection System User Bulletin #2: Relative Quantitation of Gene
Expression (PN 4303859).
Functional verification of the preloaded probes and primers inside the Low Density
Array is performed as part of the Applied Biosystems manufacturing quality control
process.
Preparing TaqMan Low Density Arrays for Use
Vent port
Fill reservoir
Main channel
Feeder channel
Reaction well
™ dye detect the amplification of user-specified
™ dye detect the amplification of the external
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