## 2 Experimental Section  
Accurate quantification and quality control (checkpoints) are important to acquire successful and reliable data, as visible in Figure 1.  
Human colorectal cancer (CRC) and normal formalin-fixed paraffin-embedded (FFPE) tissue samples were provided by the Institute of Pathology and Cytology of the Dr. Horst Schmidt Kliniken (HSK) in Wiesbaden. 8-14 serial 3 \(\upmu\)m-thick paraffin sections were kept at 60 \({}^{\circ}\)C for 10 min, deparaffinized (xylol, ethanol) and used for RNA isolation afterwards. * Checkpoint: Tissue Sample Characterization  
One section per case was HE (hematoxylin & eosin) stained for further characterization of tissue samples regarding the amount of necrosis and inflammatory cells. All tumor samples were diagnosed as low to moderately-differentiated colorectal tumors. All patients were treated with 5-fluorouracilacil (5-FU) and a total dose of 50.4 Gy. Further patient demographics and clinicopathological findings are depicted in the result part. The nucleic acid material was isolated from deparaffinized samples manually by using the Recover All Isolation Kit (Ambion) according to the manufacturer's instructions, with the addition of a 16-h proteinase K lysis step for protein degradation. To avoid DNA contamination interfering with the desired \"specific amplification\", the RNA samples were treated with RNase-free DNase. Figure 1: Experimental design (Step 1–3) of the present study, including important checkpoints () to provide quality assessment. (NC = Negative control). * Checkpoint: Optical Density (A260/280) Measurements of RNA Samples  
All nucleic acid concentrations were measured with the NanoPhotometer (Implen). The purity of nucleic acid was assessed by determining the ratio of the absorbance at 260 and 280 nm (A\({}_{260/280}\)) as basic control. An acceptable and realistic nucleic acid quality of FFPE material has an optical density (OD A\({}_{260/280}\)) ranging from 1.8 to 2.1 [6]. The RT efficiency can be affected through salt contaminations, phenols, other alcohols, and inhibitors carried over from RNA isolation process [7]. To assess the RNA integrity by electrophoretic separation, the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) or the Experion (Bio-Rad Laboratories, USA) can be used [8]. Undegraded RNA is essential for reliable gene expression analyses. In our study, the RNA integrity was observed by agarose gel electrophoresis. A disadvantage of this method may be the appearance of smears in the case of FFPE-samples. In contrast to miRNA (about \(\sim\)25 nt (nucleotide) in size) analysis which is growing in importance in oncology, mRNA (up to 10 kb) is more easily degraded by cleavage of RNAes during tissue sampling, purification and storage which also varies significantly between laboratories [9]. _Step 2: cDNA Synthesis: Reverse Transcriptase PCR (RT-PCR)_  
The mRNA strand was first reverse transcribed into its single stranded cDNA (complementary DNA) using the High Capacity cDNA Reverse Transcription Kit (ABI), containing all components necessary for quantitative conversion. The kit includes Random Primers, optimized RT Buffer, dNTP's, RNase Inhibitor and MultiScribeTM MuLV reverse transcriptase. The cDNA was then further amplified through a quantitative real-time PCR (Q-PCR). For comparative Q-PCR, normal and carcinoma tissue of the same patient were reverse transcribed. Stable \"Human RNA-Reference\" (Stratagene) was used for assay validation purposes. To confirm the absence of contaminants, a negative control (water control) should be included in the experimental design (Figure 1). A positive control (artificial template with defined concentration) can be used to detect the presence of PCR-inhibitors. To examine the quality and yield of cDNA, two primer pairs of a stable reference gene (\(\sim\)1 kb apart) can be used. The C\({}_{\mathrm{T}}\) -value of the 5' end primer pair should not exceed that of the 5' end pair by more than 1 C\({}_{\mathrm{T}}\)[9].