## 3 Experimental Results and Discussion
### Quantitative Real-Time Polymerase Chain Reaction (Q-PCR): Quality Assurance  
4.1 Validation of \(\beta\)2- Microglobulin as an Appropriate Endogenous Control Gene: Inter- Colonic Tissue Variances  
Data normalization in real-time RT-PCR is a major step in gene quantification analysis ([PERSON] _et al_., 2012; [PERSON] _et al_., 2012).  
Indeed, it is likely that mRNA levels of the desired housekeeping gene, \(\beta\)2- microglobulin, vary to such a degree that normalization becomes inaccurate and/or misleading, depending on the experimental conditions and pathohistology of investigated tissue.  
As can be seen in Figure 7, \(\beta\)2-microglobulin gene expression differs only slightly between different normal and tumor tissue samples and presents a low variability of its expression. Thus, \(\beta\)2-microglobulin can be used as an internal control gene to obtain reproducible and reliable data. _B2M_ solves the problems of variations in template starting amount and operating loading errors ([PERSON] _et al_., 2012).  
Figure 7: Comparison of RNA–\(\beta\)2- microglobulin gene expression from 25 normal and tumor colon biopsies, respectively.  
A panel of potential reference genes should be investigated on a representative number of samples to identify the most stable one required for the reliable normalization of RT-PCR data. Additionally, the Normfinder and/or geNorm software can be applied to assess gene expression stability.  
4.2 Amplicon-Specific Standard Curve Generated by Serial Dilution of a Human RNA Reference (Stratagene)  
By the application of the standard curve method, the sensitivity, variability and therefore individual real-time PCR efficiencies were determined for each applied assay used. A standard curve was derived from serial dilutions for each gene investigated (_ERCC1_, _TYMS_, _RRM1_ and _B2M_).  
The resulting standard curve is generated by plotting the log of target cDNA amount (ng) _versus_ the average threshold cycle \(\mathrm{C_{T}}\). As mentioned above, the \(\mathrm{C_{T}}\) value is the threshold cycle and presents the detected fluorescence and its geometric increase corresponding to the exponential increase of amplification in each reaction. Linear regression analysis is used to determine the slope and intercept, which respectively correspond to the amplification efficiency and number of amplicon molecules at threshold.  
The detection limit, quantification limit and quantification range were determined for every assay, as shown in Figure 8.  
Figure 8: **(A1-4)** Standard curve derived from serial dilutions of Reference RNAs (Stratagene) for different assays. **(B1-4)** The equation of the linear regression was determined for each assay of _B2M_, _ERCC1_, _RRM1_ and _TYMS_.  
As shown in Figure 8, the real-time PCR provides a large dynamic range. However, very high or very low amounts of RNA used for the following cDNA transcription led to a curtailment and loss of quality indicated by the saturated part of the semi-logarithmic standard curve depicted in Figure 8, (A1-4). To assess quality assurance, an appropriate amount of RNA/cDNA, positioned on the linear regression line, was chosen (Figure 8, B1-4). Thus, an acceptable amount of 25 ng of cDNA was used as input template amount. The standard curves (Figure 8) differ only slightly for the different genes and therefore present a low variability of their expression. For these relatively stable genes, it was possible to obtain a good reproducible standard curve on each plate. Each point represents the average of duplicated determined value and was only used with respect to a low standard deviation (not shown here).  
The table below shows the coefficient of determination (R\({}^{2}\) values), standard curve slopes and the efficiency calculated by the instrument. It indicates the  
No primer-dimers or amplified contaminated products were generated during 40 real-time amplification cycles. To check for RNA-Integrity, the Agilent 2100 Bioanalyzer or e.g., the Experion can be applied.