Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Preparing TaqMan Low Density Arrays for UseInternal StructureFigure 4-6 illustrates the Low Density Array internal structure.Fill portVent portFill reservoirMain channelFeeder channelReaction wellFigure 4-6TaqMan Low Density Array, Internal StructureDetectorsGenomic DNAContaminationThe Low Density Array permits the amplification of target and an externalendogenous control cDNA using fluorogenic 5´ nuclease assays. The assays consistof two reactions, each a complete PCR system with corresponding probe andprimers.The fluorogenic probes of the assays function as follows:• Probes labeled with the FAM dye detect the amplification of user-specifiedcDNA targets.• Probes labeled with the FAM dye detect the amplification of the externalendogenous control.TaqMan probes and primers for the cDNA targets span exon-exon junctions tominimize the contribution of contaminating genomic DNA. Performance testsdemonstrate that 5´ exonuclease assays can be run with samples containing up to10,000 copies of genomic DNA without detection of contaminants, unless otherwisespecified by Applied Biosystems.External Endogenous Control AssayNote that the commonly-used external endogenous control assay is not RNA-specificand consequently is affected by genomic DNA contamination. However, because ofthe extremely high expression level of rRNA, even gross DNA contamination has anegligible effect on the relative quantification values obtained from the Low DensityArray.Single-PlexReactionsQuality ControlThe 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 quantificationcalculations.Note: For more information on relative quantification, see ABI PRISM ® 7700Sequence Detection System User Bulletin #2: Relative Quantitation of GeneExpression (PN 4303859).Functional verification of the preloaded probes and primers inside the Low DensityArray is performed as part of the Applied Biosystems manufacturing quality controlprocess.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-11DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm