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Amplification Efficiency of TaqMan Gene Expression Assays

. Design parameters such as %GC content, Tm, and amplicon length were optimized to ensure that all of the TaqMan Gene Expression Assays have high amplification efficiency. Additionally, genome aided QC of all of the assays assures specificity for the target gene. This eliminates non-specific amplification a factor that could contribute to decreased amplification efficiency of the true target.

Testing Across a Broad Template Dilution Range

Total RNA from Stratagene Universal Human Reference RNA was reverse transcribed using random primers (Applied Biosystems High Capacity cDNA Archive kit) to generate cDNA. For each assay tested, 100 ng of cDNA was amplified with Applied Biosystems TaqMan Universal PCR Master Mix and the TaqMan Gene Expression Assay in a 50 μL volume for 40 cycles using universal cycling conditions (40 cycles of 95C for 15 seconds; 60C for 1 minute) on an ABI PRISM 7000 Sequence Detection System. This initial amplification was performed to produce sufficient template in the form of PCR product to enable the generation of a broad template dilution range.

A total of 5 μL of PCR product was run on a 4% agarose gel (NuSieve/ SeaKem) with size and quantitation standards (25 bp ladder from Invitrogen) to estimate the amount of product generated. The remaining 45 μL of PCR product was loaded on purification columns (QIAquick PCR purification kit; Qiagen) and purified according to the manufacturers directions. Bound DNA was eluted in a 100 μL volume of elution buffer. Then, 10 μL of the elute was used for picogreen staining (PicoGreen dsDNA quantitation kit, Molecular Probes) to estimate cDNA concentration.

A 6-log dilution range was generated using 10-fold serial dilutions of the target PCR product. Each of these dilutions was subjected to real-time PCR amplification as described above, using TaqMan'"/>

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