Serum outperforms plasma in small extracellular vesicle microRNA biomarker studies of adenocarcinoma of the esophagus

BACKGROUND Circulating microRNAs(miRNAs)are potential biomarkers for many diseases.However,they can originate from non-disease specific sources,such as blood cells,and compromise the investigations for miRNA biomarkers.While small extracellular vesicles(sEVs)have been suggested to provide a purer source of circulating miRNAs for biomarkers discovery,the most suitable blood sample for sEV miRNA biomarker studies has not been defined.AIM To compare the mi RNA profiles between matched serum and plasma s EV preparations to determine their suitability for biomarker studies.METHODS Matched serum and plasma samples were obtained from 10 healthy controls and10 patients with esophageal adenocarcinoma.s EV isolates were prepared from serum and plasma using Exo Quick TM and quantified using Nano Sight.RNA was extracted from s EV preparations with the mi RNeasy Serum/Plasma kit and profiled using the Taqman Openarray q PCR.The overall mi RNA content and theexpression of specific mi RNAs of reported vesicular and non-vesicular origins were compared between serum and plasma s EV preparations.The diagnostic performance of a previously identified multi-mi RNA biomarker panel for esophageal adenocarcinoma was also compared.RESULTS The overall mi RNA content was higher in plasma s EV preparations(480 mi RNAs)and contained 97.5%of the mi RNAs found in the serum s EV preparations(412 mi RNAs).The expression of commonly expressed mi RNAs was highly correlated(Spearman’s R=0.87,P<0.0001)between the plasma and serum s EV preparations,but was consistently higher in the plasma s EV preparations.Specific blood-cell mi RNAs(hsa-mi R-223-3 p,hsa-mi R-451 a,mi R-19 b-3 p,hsa-mi R-17-5 p,hsa-mi R-30 b-5 p,hsa-mi R-106 a-5 p,hsa-mi R-150-5 p and hsa-mi R-92 a-3 p)were expressed at 2.7 to 9.6 fold higher levels in the plasma s EV preparations compared to serum s EV preparations(P<0.05).In plasma s EV preparations,the percentage of protein-associated mi RNAs expressed at relatively higher levels(Ct 20-25)was greater than serum s EV preparations(50%vs 31%).While the percentage of vesicle-associated mi RNAs expressed at relatively higher levels was greater in the serum s EV preparations than plasma s EV preparations(70%vs 44%).A 5-mi RNA biomarker panel produced a higher cross validated accuracy for discriminating patients with esophageal adenocarcinoma from healthy controls using serum s EV preparations compared with plasma s EV preparations(AUROC 0.80 vs 0.54,P<0.05).CONCLUSION Although plasma s EV preparations contained more mi RNAs than serum s EV preparations,they also contained more mi RNAs from non-vesicle origins.Serum appears to be more suitable than plasma for s EV mi RNAs biomarkers studies.