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Chapter 590plasma RNA was isolated from the same healthy donors drawn in a 6 ml EDTA tube (BECTON DICKINSON). Constant factors in all the heparinase reactions were heparinase buffer (5 %u00b5l) (NEB), Protector RNAse inhibitor (2000 units, 1.25 %u00b5l) (ROCHE, Woerden, the Netherlands) supplemented with nuclease-free H2O to a total reaction volume of 50 %u00b5l. All reaction conditions were incubated for two hours at 30%u00b0C. cDNA synthesis was performed with the QIAGEN miRCURY LNA kit according to the supplied protocol, with an adjustment for RNA input. Two %u00b5l RNA and five %u00b5l H2O were replaced with seven %u00b5l RNA treated with heparinase. The cDNA was diluted 1:40 and cel-miR-39-3p was quantified using miRCURY LNA miRNA PCR Assays (QIAGEN)[33]. RNA isolated from the H630 colorectal cancer cell line spiked with cel-miR-39-3p was used as a positive control and nuclease-free water served as a non-template negative control in each experiment. The expression of cel-miR-39-3p detected after RNA isolation is correlated to the efficiency of the RNA isolation, and therefore used as a control measure for RNA quality. All samples with a cel-miR-39-3p Cq of 33 or higher were excluded for further analysis. To test the stability of synthetic cel-miR-39-3p during heparinase treatment conditions, cel-miR-39-3p was quantified using RT-qPCR after incubation for two hours at 30%u00b0C in nuclease-free H2O. miRNAs are expressed in red blood cells (RBC), lymphoid or myeloid blood cells. RBC-expressed miRNAs were reported to be increased by 20- to 30-fold in haemolysed plasma. Non%u2013RBC-associated miRNAs were not increased in haemolysed samples[36]. miR-375 and miR-141-3p are non%u2013RBC-associated miRNAs. miR-21-5p, miR-146a-5p, miR-148a-3p and miR-200c-3p are present in RBC. Therefore, the hemolytic index for each sample was defined as described previously [33]. Samples with a hemolytic index > 10 were excluded since adverse effects on non%u2013RBC-associated miRNAs cannot be excluded [37].The following miRCURY LNA PCR assays were used for miRNA quantification using ddPCR: cel-miR-39-3p (diluted 1:1), miR-21-5p, miR-375, miR-200c-3p, miR-148a3p, miR-146a-5p, miR-141-3p and miR-218-5p (QIAGEN). A thermal gradient for each primer assay was performed using colon cancer cell lines (HT29, ATCC and H630) and head and neck squamous cell carcinoma cell line VU-SCC-120. The annealing temperatures for the thermal gradient PCR ranged between 52oC and 62oC. All ddPCR procedures and reaction volumes were performed according to the manufacturer%u2019s protocol (QX200 Droplet Digital PCR System from BIO-RAD,