engleski

Performance of two different methods for microRNA extraction from exosomes of colorectal carcinoma patients

Exosomes, extracellular vesicles, and their cargo, such as microRNAs, are among the most promising emerging biomarkers of various diseases, including colorectal cancer (CRC). Since their handling and isolation are not standardised, in this study we aimed to evaluate the performance of two different methods for isolation of microRNA from CRC patients' exosomes. Exosomes and exosomal microRNA were isolated from peripheral blood of 11 CRC patients using miRCURY Exosome Serum/Plasma kit and miRNeasy Serum/Plasma Advanced kit (Qiagen) or Total Exosome isolation reagent and Total Exosome RNA and Protein isolation kit (Invitrogen). Concentration of microRNA was determined using DS- 11 spectrophotometer (Denovix), and quality of the samples was determined on Bioanalyzer 2100 (Agilent). cDNA for microRNA expression analysis was obtained using miRCURY RT LNA kit (Qiagen), while expressions of mir-103a-3p and mir-19a-3p were assessed with miRCURY LNA SYBR GREEN PCR system (Qiagen) on 7500 Real-Time PCR System (Applied Biosystems) with UniSp6 as internal control. Wilcoxon matched pairs test was used for statistical analysis in GraphPad Prism 6.01 software. There was no significant difference in RNA concentrations between the two methods (P=0.102), and both methods isolated RNAs less than 200 nt in size. ΔCt (corrected to UniSp6) for mir-103a-3p (P=0.123) and mir-19a-3p (P=0.413) was similar between the two methods. mir-19a-3p was also corrected to mir-103a-3p (usually used as reference gene) and we found no difference between the used methods (P=0.083). We concluded that both assessed methods showed similar performance and could be used for determination of microRNA expression from CRC patients' exosomes.

biomarker ; colorectal carcinoma ; exosome ; microRNA

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