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Optimization of the method for determination of selected exosomal microRNAs in liquid biopsy samples of colorectal cancer patients

Liquid biopsy offers a promising non-invasive approach for determination of the molecular profile of various tumours, including colorectal cancer (CRC). Exosomal microRNA represents one of most promising biomarkers for diagnostic screening, as well as for determining a response to therapy and evaluating disease progression and outcome. Since handling of liquid biopsy specimens is demanding and nonstandardized, in this study we aim to evaluate and optimise a method for determining selected microRNAs expression in exosome-derived microRNA samples from CRC patients. Exosomal microRNA was isolated from peripheral blood liquid biopsy samples obtained from 18 CRC patients using miRCURY Exosome Serum/Plasma kit and miRNeasy Serum/Plasma Advanced kit (Qiagen). cDNA was obtained using miRCury RT LNA kit (Qiagen) while expression of eight selected microRNAs was assessed with miRCURY LNA SYBR GREEN PCR system (Qiagen) on 7500 Real- Time PCR System (Applied Biosystems) with UniSp6 as internal control. Initial examination of expression of three microRNAs with four different starting sample volumes showed satisfactory results with the starting microRNA volume of 2 µl. miR-103a-3p was shown to be the best reference microRNA candidate, with Ct values ranging from 27.40 to 36.05 and ΔCt (corrected to UniSp6) from 13.36 to 22.31, since miR-1228-3p and miR-520d-3p were not detected. miR-125-3p was detected in three samples with Ct ranging from 33.58 to 35.86, and ΔCt from 20.01 to 22.40 ; miR-193a-3p was detected in seven samples with Ct ranging from 36.13 to 42.17, and ΔCt from 21.15 to 27.70 ; miR- 210-3p was detected in 14 samples with Ct ranging from 31.15 to 42.91 and ΔCt from 17.71 to 29.18, whereas miR-19a-3p and miR-92a-3p were detected in all samples with Ct ranging from 32.20 to 32.67 and 23.87 to 31.96, and ΔCt ranging from 9.90 to 16.74 and 10.35 to 16.97, respectively. The described optimisation of the method for the detection of selected exosomal microRNAs in samples obtained by the peripheral blood biopsy will contribute to the further profiling of CRC utilizing liquid biopsy through comparison with expression in tumour tissue samples.

colorectal cancer ; exosome ; liquid biopsy ; microRNA

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