15283-B-Blokker

128 Chapter 7 Handling and processing RNA may have major impact on RNA quality and therefore on the outcomes of the study. Inconsistency in executor and work performance, and contamination of RNA (with RNase or other genetic material) must be avoided when working with RNA. 186,198 The experiments were therefore performed by one qualified and experienced researcher (AvL). To all RT-qPCR assays a dilution series was added to assess the performance of both the assay and the researcher’s skills. The efficiency numbers (table 1) show that the RT-qPCR assays were all well performed, although the efficiency decreases with amplicon size. The observed (parallel) increase of Cq values in all samples (i.e. post-mortem as well as fresh frozen; figure 2) in relation to the increasing amplicon size may thus be due to decreasing PCR efficiency, rather than increased RNA degradation. The integrity of post-mortem tissues is subject to various factors. 188,199 Our results suggest that RNA in MIA tissue is less degraded than RNA derived from CA tissue. However, in this study CA was always performed after MIA, so the longer PMI is probably the explanation for this finding. Yet it is possible that differences intrinsic to MIA and CA play a role as well. 181,183,200 More extensive exposure of tissues to air, which occurs during conventional autopsies, could also have a negative influence on tissue quality. It is known that vacuum sealing of tissue specimens has a beneficial effect on RNA preservation. 201 Regarding patient conditions, our analyses show seemingly contradicting results: whereas hypoxia, BMI, hypertension, and dyslipidemia do not seem to have an effect on RIN values, hypoxia, fever and diabetes did not show significant differences between Cq values. The RNA quality according to the RIN values was significantly lower in cases with reported fever and diabetes, the RNA quality according to the Cq values was significantly lower in cases with dyslipidemia and a Body Mass Index (BMI) of >25, and in cases with hypertension, the Cq values differed significantly for only some GAPDH base pair lengths. The inconsistent results are probably due to the low number of samples from different cases. Agonal factors, such as hypoxia and fever are known to have a negative influence on RNA quality and can lead to inhomogeneous RNA samples in different tissue types. 202 To our knowledge, the individual influence of hypertension, dyslipidemia and diabetes mellitus on post mortem tissue quality is unknown. Together however, these factors are described as "the metabolic syndrome", which is associated with obesity. 203 The few differences found in RNA quality might therefore be explained by the confounder obesity. Obesity is in fact correlated with post mortem tissue quality, as it causes temperature insulation by fatty tissue, leading to higher core temperatures that are maintained longer. These higher temperatures enhance decomposition (autolysis