Nanodrop lite nd lite pr

Total RNA from macroscopically dissected superficial tumorous tissues of lesions with a minimal size of approximately 5 mm and remnant surrounding liver tissues with smaller lesions (smaller than 5 mm) was separately isolated using peqGOLD RNAPure (30-1010, Peqlab, Erlangen, Germany), quantified at 260 and 280 nm with Nanodrop Lite (ND-LITE-PR, Thermo Fisher Scientific, Waltham, MA, USA) and reverse transcription into cDNA by Omniscript RT Kit (205113, Qiagen, Hilden, Germany) was performed as described recently [7 (link)]. Relative quantitative gene expression was measured via real-time PCR using a QuantStudio 6 Flex Real-Time PCR System with Real-Time PCR Software v1.3 (Applied Biosystems, Foster City, CA, USA). Target gene expression was normalised to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression as internal standard and calculated as fold induction in comparison to controls as indicated. All primer sequences used for qPCR (Eurofins Genomics, Louisville, KY, USA) are given in Supplementary Table S3.

To avoid the accumulation of single nucleotide variants during the passage process, viral DNA for MGI platform sequencing was extracted directly from specific tissues. The first-generation culture of porcine bone macrophages was used to prepare extracellular virions for Nanopore sequencing. The QIAamp DNA Mini Kit (51304, Qiagen, Germany) was used for extraction, and the extracted viral DNA was purified using the AMPure XP Purification Kit (A63880, Beckman Coulter, USA). The quality and concentration were evaluated using Nanodrop Lite (ND-LITE-PR, ThermoFisher, USA) and Qubit 4.0 Fluorometer (Q33238, Invitrogen, USA). All these procedures were performed according to the manufacturer's instructions.

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.