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

Chapter 4Operating the InstrumentPreparing TaqMan Low Density Arrays for UseAbout the Low Density ArraysThe TaqMan Low Density Arrays are research tools for profiling gene expressionusing the Comparative C T Method of relative quantification. The Low Density Arrayevaluates from one to eight cDNA samples or controls generated from human totalRNA in a two-step RT-PCR experiment.How It WorksLow DensityArrayComponentsThe Low Density Array functions as an array of reaction vessels for thePCR/sequence detection step. Typically, the wells of the Low Density Array containApplied Biosystems fluorogenic 5´ nuclease assays (TaqMan ® reagents; see the notebelow) that detect the real-time amplification of user-specified targets. Relativelevels of gene expression are determined from the fluorescence data generated duringPCR using the Applied Biosystems 7900HT Fast Real-Time PCR System RelativeQuantification software.Note: In this document, the term TaqMan reagents refers to 5´ exonuclease assayreagents that feature Applied Biosystems TaqMan ® primers and probes (includesApplied Biosystems PDARs, TaqMan ® SNP Genotyping and Gene ExpressionAssays, Custom TaqMan ® SNP Genotyping and Gene Expression Assays, and anycustom TaqMan reagents.)The Low Density Array (Figure 4-5) acts as a vessel for real-time PCR. It consists ofa series of 384 interconnected wells divided into eight sets of assays. Each setprovides a user-specified number of replicates. Each well contains dried AppliedBiosystems TaqMan primers and probes for one mRNA target.Fill reservoir (1 of 8)A reservoir for the cDNA sample orcontrol before it is centrifugallyloaded into the wellsFill consumableThe segment of the Low DensityArray that contains the eight fillreservoirs. (After the Low DensityArray is centrifuged and sealed, thefill consumable is trimmed off.)GR2156aBarcode Provides coded access toLow Density Array configurationdatabasesFigure 4-5The TaqMan Low Density Array4-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm