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Plasma microRNAs in oesophageal and gastric cancer895Table 1. Literature background for miRNA selection in oesophageal and gastric cancer. Abbreviations: H, high expression; L, low expression; ESCC, oesophageal squamous cell cancer; EAC, oesophageal adenocarcinoma; GC, gastric cancer; C, circulating; T, tissue; HR, hazard ratio; p, p value, all p values lower than 0.05 were noted as <0,05; *, HR missing; %u2193, shorter OS; %u2191, longer OS; ** for this study relation with PFS was reported, no relation with OS.miRNA No. studies HR for OS(95% CI) Relation miRNAOSp-value No. pts, histologySpecimen RefmiR-21 (H) 511.87*1.4-2.6 %u2193%u2193< 0.05< 0.05504 ESCC/EAC38 ESCCTCC[25][17]miR-375 (H) 690.551.690.4-0.71.5%u20132.0%u2191%u2193< 0.05< 0.050.05723 ESCC1230 ESCCTCTC[21][21][30]miR-148a (H/L)miR-148a (L)11**%u2193(H:EAC)%u2193(L: SCC)%u2193< 0.05< 0.0545 ESCC/EAC49 ESCCTT[10][27]miR-141(L) 11**%u2193%u2193< 0.05< 0.0595 GC30 GCTT[18][31]miR-200c-3p (H) 11111*2.244.011.67*1.1-4.62.8-10.01.1-2.4%u2191%u2193%u2193%u2193%u2193**< 0.05< 0.05< 0.05< 0.05< 0.0551 GC52 GC98 GC157 ESCC64 ESCCT/CCCCC[16][13][15][30][24]miR-146a(L) 312.601.531.63-4.131.06-2.26%u2193%u2193<0.05<0.05213 GC90 GCTT[12][20]miR-218 (L) 113.163.191.06-9.401.55-8.37%u2193%u2193<0.05<0.0568 GC112 GCCT[19][32]RNA isolation, heparinase treatment, RT-qPCR, sample quality and droplet digital PCRRNA isolation of heparinized plasma samples from the included patients was performed using the miRNeasy Serum/ Plasma Advanced Kit (QIAGEN, Venlo, the Netherlands) according to the manufacturer`s protocol, with adjustments as described previously [33] and stored at -80%u00b0C until further analysis. Heparin contamination interferes with the reverse transcriptase polymerase chain reaction [34]. Therefore the RNA samples were treated with Bacteroides heparinase I (NEB, Leiden, the Netherlands, 12.000 U/ml) [35]. In order to optimize the heparinase treatment of plasma RNA, RNA from plasma samples of 3 healthy donors was isolated and treated with heparinase under different conditions. Different volumes of isolated plasma RNA (10, 20 or 30 %u00b5l) were incubates with different quantities of heparinase I (6, 12 or 18U). Synthetic cel-miR-39-3p (spiked during the RNA isolation procedure) was quantified with RT-qPCR as read-out for heparinase treatment efficiency in these samples. As a positive control for this experiment
