Figure 5
Comparison of TaqMan® gene expression pattern using ΔC T method. (Am = Ambion protocol; FP = FFPE cells; + = modified protocols with incubation in Proteinase K buffer at 70°C for 20 minutes; and SN = snap frozen cells. Not all assays produced products, e.g. HLA_A in UPMM.) These ΔCT data was generated from the CTs shown in Figure 3. A Theoretical ΔCT was calculated for each chart based on equilibrating the results for any variation in input cDNA. In panel a, ΔCT = CT_UPMM - CT_GEMM. The Theoretical ΔCT of UPMM-GEMM was 0 [= Log2(20 ng/20 ng)] given identical input quantities (20 ng) were used in each system. In panel b, ΔCT = CT_UPMM - CT_PreAmp. In panel c, ΔCT = CT_GEMM - CT_PreAmp. The Theoretical ΔCT in panel b and c was 5.68 [= Log2(1024 ng/20 ng)], which was calculated based on an input of 1024 ng of cDNA for the TaqMan® real time PCR component of pre-amplification process. This quantity was generated from an initial 1 ng subjected to 10 cycles of pre-amplification with a 100% efficiency and no bias introduced from the PreAmp. The relevant Theoretical ΔCT is plotted on each chart as a reference point for measuring the actual detected ΔCT against the theoretically optimal ΔCT. The benefit of GEMM over UPMM was evident as amplicon size increased (Panel a). A comparison of UPMM and PreAmp showed a similar pattern (Panel b). However, PreAmp results generally correlated with GEMM regardless of amplicon size for the series of 8 assays analysed (Panel c).