15283-B-Blokker

126 Chapter 7 Figure 1-B shows the median RIN values and interquartile range per sample type and tissue type. For each tissue type the RIN values of fresh frozen samples were significantly higher than those in post-mortem samples (P<0.001, unpaired Mann- Whitney test, significance level 0.05). No significant differences in RIN value per tissue type were found between MIA and CA. The morphological factor autolysis adversely influenced RNA integrity (P < 0.001). Of the patient related agonal factors, hypoxia, BMI, hypertension and dyslipidemia did not influence RNA integrity, but fever and diabetes did have an adverse effect on the RIN value in post-mortem samples (both P = 0.006, unpaired Mann-Whitney test, significance level 0.05). RT-qPCR RT-qPCR was performed on all 218 samples and Cq values could be established for 216 samples. In one case the RNA yield was too low, in the other case the Cq value could not be measured due to technical failure. These samples were excluded from the analyses concerning Cq values. Figure 1-C shows that median Cq values are lowest in fresh frozen samples for the GAPDH 71 bp assay. The Cq values in CA samples are highest. Figure 1-D show the Cq values per tissue type. Liver tissues have the highest Cq values for all sample types. Figure 2 shows the Cq values of all GAPDH amplicon sizes per sample type. The difference in Cq values between fresh frozen and post-mortem samples (MIA and CA) is significant for all GAPDH amplicon sizes (P< 0.001, Paired Kruskal-Wallis test, significance level 0,00833). The difference in Cq values between MIA and CA samples is not significant for all GAPDH amplicon sizes, as shown in table 6. When the Cq values were stratified per tissue type, there were no significant differences between MIA and CA samples. The RNA integrity of all samples decreases with increasing post-mortem interval (see figure 3-A). Figure 3-B shows the RIN values of the three different tissue types per PMI. The fresh frozen samples are represented by PMI category “0”. The RIN value in this category was significantly higher than in all other categories, but there were no significant differences in RIN values between PMI categories 1 to 5. According to figure 3-C, the GAPDH 71 bp assay Cq values increase with PMI (i.e. decrease of GAPDH expression level). Figure 3D shows the GAPDH 71 bp assay Cq values per tissue type per PMI. Cq values in PMI category 2 are significantly higher than those in PMI category 1.