Quantifying miRNA Biomarkers in sEVs for EBL

To quantify the levels of miRNA biomarker candidates for EBL in sEVs, we used three BLV-uninfected (Nos. 1–3) and three EBL cattle (Nos. 4–6) (Supplementary Table S1). Total RNA was extracted from the sEVs as described in the microarray analysis section. The concentration and quality of the extracted total RNA were estimated using a NanoDrop Lite (ND-LITE-PR, Thermo Fisher Scientific). We synthesized cDNA from 6 μL of 5 ng/μL of extracted total RNA using miRCURY LNA RT Kit (339340, Qiagen) according to the manufacturer’s instructions and used it for qPCR. Quantification of the miRNA biomarker candidates was performed using the miRCURY LNA SYBR Green PCR Kit (339346, Qiagen). Primers for gga-miR-17-5p (YP00205960, Qiagen), hsa-miR-24-3p (YP00204260, Qiagen), cfa-miR-210 (YP02119434, Qiagen), and hsa-miR-92a-3p (YP00204258, Qiagen) were contained in miRCURY LNA PCR Assay components (339306, Qiagen). The detailed sequence information of primers is provided in Supplementary Table S3. For qPCR, the cDNA was diluted at 1:30 by adding RNase-free water, and 3 μL of diluted cDNA was used in a total reaction volume of 10 μL. We performed qPCR as described previously [30 (link)]. Because suitable internal control miRNAs encapsulated in bovine blood sEVs have not been identified yet, we evaluated miRNA amounts in blood sEVs using Ct values.