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Chapter 592control group was 28.26 %u00b1 0.01 SEM (pink diamonds, Figure 1). Twenty %u00b5l RNA incubated with 6 U heparinase (28.96 %u00b1 0.08 SEM) and 30 %u00b5l RNA incubated with 12 U heparinase (28.93 %u00b1 0.14 SEM) were the most efficient volumes for celmiR-39-3p detection (Figure 1). Thirty %u00b5l RNA incubated with 12 U heparinase was selected for this study. To define if cel-miR-39-3p is degraded during the heparinase treatment, it was incubated for 2 hours at 30oC in H2O. No significant effect was observed when a similar concentration of cel-miR-39-3p as spiked-in during the RNA isolation was incubated for 2 hours at 300C in H2O (Figure 1). CelmiR-39-3p was measurable in duplicate in 52 (82,5%) baseline samples and in 30 (56,6%) follow-up samples. Twenty-seven samples were excluded (15 (28,8%) baseline and 12 (40%) follow-up) due to a low (Cq %u226433) cel-miR-39-3p expression (Figure 2). Five samples were excluded (1 (1,9%) baseline and 4 (22,2%) followup) based on a high (>10) hemolytic index. As a result, 36 (69,2%) of the 52 baseline samples (10GC;19EAC;8ESCC) and 14 (46,7%) of the 30 follow-up samples (7GC;5EAC;2ESCC) were included for further analysis (Supplementary Figure S1). Baseline characteristics of the patients with measurable plasma samples that complied with the quality criteria are described in Table 2.(a) (b)Figure 1. (a) The effect of different heparinase quantities (Units, U) in combination with different RNA volumes on the detection of cel-miR-39-3p by RT-qPCR. Each point in the graph represents the raw Cq value of duplicate RT-qPCR measurements of cel-miR-39-3p in plasma RNA isolated from three different healthy donors. RNA input is shown on the x-axis and raw Cq value of cel-miR-39-3p is shown on the y-axis; (b) The detection of 20 fM cel-miR-39-3p in water by RT-qPCR after different incubation times. The H2O samples were not treated with heparinase. Each point in the graph represents the raw Cq value on the Y-axis of duplicate RT-qPCR measurements from two different samples on the x-axis.
