## 3 Experimental Results and Discussion
### Optical Density (A\({}_{260/280}\)) of RNA Samples  
Variations in quantity and quality of samples due to partial degradation or the presence of contaminations can influence the data outcome. Thus, it is very important to work under reproducible conditions for every sample. Figure 5: Histomorphological Characterization. (**A**) Samples (n = 50) at stage of diagnosis and (**B**) after neoadjuvant radiochemotherapy, used for further analytical purposes. Most samples contain a large number of viable tumor cells and only a low to moderate percentage of necrosis, lymphocytic and granulocytic infiltrations. Table 4 presents the overall purity and thus the quality of all isolated RNA samples collected before and after treatment of 25 patients. As a basic control, the purity of the nucleic acid was determined according to the ratio of the absorbance at 260 nm and 280 nm (A\({}_{260/280}\)), ranging in this study from 1.8 to 2.1. To investigate the RNA, samples can be electrophoretically separated by applications from both Bioanalyzer and Experion. All absorbance values obtained were in an acceptable range. Therefore, all samples remained included in this study. The diagrams (Figure 6) also illustrate an overall poorer RNA quality in tumor tissue compared to corresponding normal tissue. The conventional Reference RNA (Stratagene), used to assess the efficiency of the Q-PCR system by the standard dilution curve method, showed the best stable quality even after several repeats, indicated by an OD\({}_{260/280}\) of 1.9 (Table 4). The sensitivity and specificity of the analysis of conventional RNA tumor samples is limited not only by stromal contamination and by heterogeneity within the cancer, but also by the state of the isolated RNA material. RNA is much more labile than DNA, due to high tissue concentrations of endogenous lytic enzymes, and is substantially degraded by formalin fixation and paraffin embedding. Handling and storage of RNA at room temperature can also influence degradation. Extraction of RNA is a long process, often undertaken in the presence of contaminants and ribonucleases that may degrade the nucleic acid. Storage processes can also be a problem, especially for gene analysis. Through the application of shock frozen tissue instead of FFPE samples, as used by some other diagnostic laboratories, can decrease what would otherwise be a rapid degradation or contamination by RNAase of the environment, especially in the case of RNA isolation. \begin{table}
\begin{tabular}{|l|c|c|c|} \hline
**Nucleic acid** & **OD\({}_{\text{260/280}}\) (mean)** & **OD\({}_{\text{260/280}}\) (median)** & **OD\({}_{\text{260/280}}\) (standard deviation)** \\ \hline RNA & 1.9 & 1.8 & 0.283 \\ \hline RNA Reference (Stratagene) & 1.9 & 1.9 & 0 \\ \hline \end{tabular}
\end{table} 
Table 4: Optical density (OD\({}_{\text{260/280}}\)) of nucleic acid including the purchased RNA Reference (Stratagene). Figure 6: RNA purity characterized by optical density (OD\({}_{\text{260/280}}\)) measurements. By considering all FFPE samples, the highest purity was obtained in RNA samples of normal individual colonic tissue of the patient as compared to corresponding tumor tissue. * aT = tumor tissue sample before treatment; bT = tumor tissue sample after treatment; bN = normal tissue sample after treatment. The normal tissue used within this study always came from the closest non-involved area of the patient whose tumor was being studied. A poorer purity of nucleic acid in the colon adenocarcinoma over the normal tissue was demonstrated by optical density measurements. This was possible due to a high level of enzyme activity in neoplastic tissue above those found in normal tissues from which they originated, and was carried out in accordance with a study performed by [PERSON] _et al_. [PERSON] _et al_. (2012).