## 3 Experimental Results and Discussion
### Quantitative Real-Time Polymerase Chain Reaction (Q-PCR): Quality Assurance
#### 2.4.3 Intra- and Inter-Assay Variation: Reproducibility of the Real-Time PCR
It was established that the quality of the isolated RNA from different samples varied only slightly. Nevertheless, this variation could have an influence on the resulting measured C\({}_{\mathrm{T}}\) values. Therefore, a stable Reference RNA such as the one available from Stratagene has found its application in some other routine diagnostic laboratories. However, does this RNA Reference really substitute the individual normal RNA isolated from the colonic tissue of the respective patient where the tumor was also found? In order to use one Reference RNA (obtained from RNA isolated from different cell culture cells) suitable for all tumor samples investigated, it must be assumed that there is no individual expression level variation of the investigated RNA species between different patients. Because this assumption is difficult to prove, normal tissue from the investigated patients was used. Figure 12: Different relative gene expression profiles for \(ERCC1\), \(RRM1\) and \(TYMS\)\(ERCC1\) and \(RRM1\) show similar expression pattern. It is visible that those patients with an increased \(TYMS\) expression showed no or only little tumor response (regression grade 0–1) to 5-FU chemotherapy. As normal tissue samples are not always available in all cases in which tumor tissue was obtained, the application of a standard Reference RNA would be an alternative. It should be emphasized that tumor tissue and no single isolated cells were utilized in this study. Therefore, the normal tissue was used as calibrator because otherwise it is difficult to attribute all of the elevated activity to the actual neoplastic cell because some normal cells may be included in the tumor mass. \({}^{\text{-}\Delta\text{ACT}}\) Comparative Gene Expression of Individual Tumor Tissue of Each Investigated Patient  
The relative gene expression was determined by using the 2-\({}^{\text{-}\Delta\text{ACT}}\) comparative method. All data were normalized to _$2-microglobulin_ (_$2M_) mRNA content. Comparative RNA expression analysis in tumor _versus_ normal cells (calibrator) was performed as follows: \begin{tabular}{l l}  & Tumor tissue: \(\Delta\text{C}_{\text{T}}=\text{C}_{\text{T}}\left(\text{target}\right)-\text{C}_ {\text{T}}\left(\beta 2M\right)\) \\  & Normal tissue: \(\Delta\text{C}_{\text{T}}=\text{C}_{\text{T}}\left(\text{target}\right)-\text{C}_ {\text{T}}\left(\beta 2M\right)\) \\  & \(\Delta\text{C}_{\text{T}}=\Delta\text{C}_{\text{T}}\text{(tumor tissue)}-\Delta\text{C}_{\text{T}}\text{(normal tissue)}\) \\  & Ratio = 2-\({}^{\text{-}\Delta\text{ACT}}\) \\  & \\  & \\  & \\  & \\ \end{tabular}   
Example: \begin{tabular}{l l}  & Tumor tissue: \(\Delta\text{C}_{\text{T}}=29.304-25.211=4.09\) \\  & Normal tissue: \(\Delta\text{C}_{\text{T}}=29.300-24.860=4.44\) \\  & \\  & \\  & \\  & Ratio = 2-\({}^{\text{-}(-0.35)}=1.27\) \\ \end{tabular}   
The \(\Delta\text{C}_{\text{T}}\) value was calculated by respectively subtracting the average Ct value of the _$2M_ RNA from the average \(\text{C}_{\text{T}}\) value of the normal tissue and of the tumor tissue. The \(\Delta\text{C}_{\text{T}}\) value was calculated by subtracting the \(\Delta\text{C}_{\text{T}}\) value for the normal cells from the \(\Delta\text{C}_{\text{T}}\) value of the tumor cells [13]. Fold of enrichment values was determined according to the ratio 2-\({}^{\text{-}\Delta\text{ACT}}\) (see example). A correlation between the regression grade and the expression level of _TYMS_ was found (Figure 13), including in one female patient who died from this cancer, who had a very high _TYMS_ expression level and showed no tumor response to 5-FU radiochemotherapy. **Figure 13.** One factor analysis of _TYMS_ according to the regression grade (after Dworak) [11]. The t-test also reveals a significant correlation between _TYMS_ and the grade of regression by clear separated cycles.