15283-B-Blokker

124 Chapter 7 5 fresh frozen samples and 9 MIA samples contained so little tissue, that it was all needed for the RNA isolation and no histologic slide could be made. For 3 fresh frozen samples, 2 MIA samples and 6 CA samples the necrosis could not be scored, due to freezing artifacts rendering the interpretation of the morphology uncertain. These cases were registered as ‘no scoring possible’ (n/s), due to severe autolysis. cDNA synthesis and RT-qPCR Some smaller tissue samples did not yield the required concentration of 100 ng/μl RNA for cDNA synthesis. In order to achieve a concentration of 100 ng/μl total RNA per sample, a volume containing 1 μg of RNA from all samples (to assure equal treatment of all samples) was transferred to another test tube and air dried by Vacuspin (SpeedVac AES1010, Savant, USA) for 30 minutes at 45 $ C. 10 μl of RNase free water was then added to reconstitute the RNA samples to the required 100 ng/μl total RNA. cDNA synthesis of all samples, including a positive control (MCF7 cell line RNA) and a no template control (NTC), was performed as previously described. 177 Three pools of randomly picked cDNA samples were made by taking 1μl of cDNA of 10 fresh frozen (pool 1), 10 MIA (pool 2) and 10 CA (pool 3) samples and diluting them twenty times in water. A fourfold dilution series was made of the initial pool samples to create a sensitivity curve. This additional step, consisting of fifteen samples in total, was added to each PCR assay to assess the efficiency of the assays. To determine the quantity (degradation in post-mortem samples compared to the fresh frozen samples) and quality (acceptable length of RNA fragments for demanding downstream RNA based genomic techniques), quantitative real time polymerase chain reaction (RT-qPCR) with the GAPDH amplicon size assay (table 1), as previously described by Viertler et al, 192 was performed on all patient and control samples. Data analysis Differences in RIN values and Cq values, with respect to tissue types (heart, kidney and liver); sample types (fresh frozen, MIA and CA); PMI; clinical/patient related data and morphology scores were analyzed in SPSS (IBM SPSS Statistics, version 21.0) using the Mann-Whitney U test and the Paired Kruskal-Wallis test. Since the Cq values were measured for 6 GAPDH base pairs per tissue sample, multiple testing needed to be applied. 193,194 Therefore, the usual significance level of P=0.05 was divided by 6, resulting in a significance level of P=0.00833, which was used for the analyses of RT-qPCR outcomes.