## 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  
The precision and reproducibility of the real-time PCR to further understand the limitations within the system was examined by intra- (replicates in the same PCR run) and inter-assay (replicates in separate kinetic PCR runs) amplification. As shown in Figure 10, the intra-assay precision was determined in eight repeats of reverse transcription and measured as duplicates. Determination of variation was done in 25 ng transcribed total Human RNA Reference (ABI). The coefficient of variation was determined as depicted in Table 6. \begin{table}
\begin{tabular}{|c|c|c|c|c|} \hline
**Assay** & **CP\({}_{\text{mean}}\)** & **CP\({}_{\text{standard deviation}}\)** & **Intra-Assay Variation (n = 8) CV [\%]** & **Inter-Assay Variation CV [\%]** \\ \hline \(\beta\)-_2m_ & 20.87 & \(\pm\)0.11 & 0.5 & \\ \hline _ERCC1_ & 23.42 & \(\pm\)0.18 & 0.8 & \\ \hline _RRM1_ & 23.9 & \(\pm\)0.17 & 0.7 & \(\leq\)25 \\ \hline _TS_ & 23.87 & \(\pm\)0.15 & 0.6 & \\ \hline \end{tabular}
\end{table} 
Table 6: Assay variations are based on CP variation and expressed as CP\({}_{\text{mean}}\), CP\({}_{\text{standard deviation}}\) and CV (coefficient of variation). Figure 10: Determination of intra-assay variation in 25 ng reverse transcribed total RNA. Figure 9: Confirmation of primer specificity after cDNA-amplification. Inter (CV = \(<\)1%) and intra (CV \(\leq\) 25%) assay variations are in an acceptable and realistic range according to [PERSON] _et al_. [7]. 4.4 \(\Delta\)C\({}_{\rm T}\) Values of Individual Normal Tissue of the Patient as Calibrator: Assay Variability between Different Individuals. As shown below (Figure 11), the \(\Delta\)C\({}_{\rm T}\) values were determined for _ERCC1_, _RRM1_ and _TYMS_ mRNA expression profiles of the respective normal individual tissue collected from 25 patients. The \(\Delta\)C\({}_{\rm T}\) value is the difference between the target gene and endogenous control, described as:  
As depicted in Figure 12, no significant correlations between the obtained \(\Delta\)C\({}_{\rm T}\) values, the age (Chi\({}^{2}\)-test, \(p\) = 0.358) and sex (Chi\({}^{2}\)-test, \(p\) = 0.274) were evaluated. _TYMS_ indicates a more variable gene expression among different individual patients as compared to _ERCC1_ and _RRM1_. Figure 11: Differences in gene expression are not correlated with age or sex (1 = female; 2 = male). _ERCC1_, _TYMS and RRM1_ assays show the same pattern of variances in gene expression. The _ERCC1_ and RRM1 expression profile seems to be more significantly correlated as compared to _TYMS. TYMS_ is a little bit more variable in its gene expression among different individuals than _ERCC1_ and _RRM1_. For two patients no fluorescence signals were detected, although the isolated RNA quality was in an acceptable range. One possible explanation might be that the RNA may have contained mutations and therefore the primers could not bind properly and the RNA fragment was not amplified. Individual changes in gene expression pattern of normal colonic tissue were detected. One can hypothesize that a significant increase in, _i.e._, \(ERCC1\) gene expression in normal tissue over the tumor tissue would be the best indicator for successful treatment with platinum-based drugs, because of reduced systemic side effects. Thus, the high activity of repair enzymes (_i.e._, \(ERCC1\)) in normal tissue could prevent damage to the DNA and cut out the chemotherapeutic agent rapidly before inducing apoptosis. However, a decreased expression of this repair gene and therefore enzyme in tumor tissue would also be a benefit for the patient. In that case the chemotherapeutic agent can bind and damage the nucleic acid and initiate apoptosis without being cut out directly. Thus, the RNA expression in the normal individual may in addition be used as a predictive value of the effect and outcome of chemotherapeutic drugs. In this study, the normal colonic tissue of the patient was used as calibrator.