## 2 Experimental Section
_Step 3: Quantitative Real-Time PCR (Q-PCR)_  
Different technologies exist to investigate the transcriptome on RNA level. For instance, the microarray analysis may examine the gene expression of thousands of genes (up to 20,000) in a short time but has a high variability and insufficient sensitivity. Another technique to analyze the mRNA expression level is the RT-PCR by HPLC separation and UV detection or high resolution gel electrophoresis followed by densitometric analysis. In addition, gene quantification can be determined by LightCycler (Roche) or ABI 7500 technologies using \"hybridization probes\" [9]. The latter one with up to a 96-well standard format was used in this study. The best-known probe system, which has also been used in the present study, is ABI's TaqMan [7]. The TaqMan gene expression assay is a ready to use 5'-3' _Taq_ polymerase assay containing TaqMan(r) dye-labeled probes (FAM/TAMRA) and desired primers as listed in Table 1.  
In contrast to the available SYBR-green based detection, TaqMan probes detect specific amplification products only. Probe hydrolysis separates fluorophore and quencher, which results in an increased fluorescence signal which can be measured. This method enables fluorescent monitoring of amplified DNA in real time, at which a threshold amount of amplicon cDNA is produced. The cycle threshold (C\({}_{\mathrm{T}}\)) value describes the point at which the fluorescence crosses the threshold (Figure 2). According to [PERSON], the Q-PCR reaction can be divided into four characteristic phases (Figure 2) [7]. The amount of amplified target is directly proportional to the input amount of target during the exponential phase of PCR amplification. By using the C\({}_{\mathrm{T}}\) data, the gene mRNA expression can be explored. The output data of quantitative PCR are shown and discussed in detail later. _82-microglobulin (_82M_), a component of the MHC \begin{table}
\begin{tabular}{c c c c c} \hline
**Gene** & **Gene** & **Assay** & **Location** & **Amplicon-Length** \\  & **Symbol** & **Assay** & **Location** & **Amplicon-Length** \\ \hline _Thymidylate-Synthae_ & _TYMS_ & Hs00426586\_ml & Exon 6-7 & 60 bp \\ _\(\beta\)-2-Microglobulin_ & _\(\beta\)-2m_ & Hs00187842\_ml & Exon 1-2 & 64 bp \\ _Ribonucleotide Reductase M1_ & _RRM1_ & Hs01040705\_ml & Exon 8-9 & 57 bp \\ _Excision Repair Cross-Complementing Repair Gene, Complementation group 1_ & _ERCC1_ & Hs01012158\_ml & Exon 3-4 & 55 bp \\ \hline \end{tabular}
\end{table} 
Table 1: Highly purified salt-free primers for gene targeting (ABI). Figure 2: Sigmoidal-shaped amplification plot showing increases in fluorescence signal (phase 1–4). Fluorescence signal _versus_ cycle number (C\({}_{\mathrm{T}}\)). _Delta Rn_ (= (Rn\({}^{+}\)) - (Rn\({}^{-}\))) indicates the magnitude of the signal intensity generated by a given set of PCR conditions. A decreased C\({}_{\mathrm{T}}\) value (x axis) indicates a higher amount of starting template. The Rn\({}^{-}\) value is obtained as a ratio for a PCR without template (the no-template control) and the Rn\({}^{+}\) value is the ratio of FAM fluorescence intensity to the fluorescence intensity of the passive reference dye (ROX) included in the reaction mixture for a PCR with template [7]. (Major Histocompatibility Complex) class I molecule, was used as endogenous control and normalization factor for the RNA concentration of each sample. Thus, in this experiment, _$2-microglobulin_ gene content should remain constant relative to total nucleic acid. For a comparative gene expression analysis, the specific individual normal tissue of the patient was used as calibrator. * Checkpoint: Confirmation of Primer Specificity and Amplified PCR End Products  
The real-time PCR end product was later documented by 4% ethidium bromide agarose gel staining to ensure proper target amplification, which results in a single band with the desired length (Table 1). Generally, the typical dissociation curve is applied to detect nonspecific amplification in cDNA samples.