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Amplification Efficiency of TaqMan Gene Expression Assays

p> Universal PCR Master Mix without AmpErase UNG on an ABI PRISM 7900HT Sequence Detection System. The CT values obtained over this 6-log dilution range were plotted against cDNA concentration (Figure 1). Dilutions with a CT higher than 35 were not included in the results because CT values greater than 35 approach the sensitivity limits of the real-time PCR detection system. Amplification efficiency was calculated from the slope of this graph using the equation:

Equation 3

For example, if the slope of the line is -3.33, then the amplification efficiency is:

Use of the Comparative CT Method for Determination of Fold Change in Gene Expression

Stratagene Universal Human Reference RNA (100 ng/μL) was reverse transcribed as previously described to obtain cDNA. The cDNA was diluted in a 100-fold dilution range to obtain a dilution series with known amounts of cDNA. A constant amount of cDNA reverse transcribed from a nonhuman transcript (not detected in the Stratagene RNA pool) was used as a control to measure fold change. This transcript was made by T7 transcription of a 227 bp fragment from the Hepatitis C virus 5' UTR inserted into a TOPO-TA plasmid. Each of the Universal Human Reference cDNA dilutions was subjected to real-time PCR amplification as described above, using TaqMan Universal PCR Master Mix without AmpErase UNG on an ABI PRISM 7900HT Sequence Detection System. The relative quantities of diluted target cDNA vs. Hepatitis C cDNA were determined by either the nnCT method (Applied Biosystems User Bulletin #23) or by inserting the pre-determined efficiency values for target and endogenous control into the equation:

Equation 4

For derivation of the above equation, see Li
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