Taqman microrna specific primers

Spin column-based isolation of exosomal RNA from 1mL of urine was performed using exoRNeasy Serum/Plasma midi kits (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. There was no DNA contamination and the integrity of the RNA was confirmed with agarose gel electrophoresis and Agilent 2100 Bioanalyzer. RNA was stored at -80°C until use. BKV microRNA expression was assessed by quantitative real-time reverse transcriptase PCR (qRT-PCR) using human TaqMan microRNA assays (Applied Biosystems, Foster City, CA). Reactions using 3 μL of RNA were performed with TaqMan microRNA Reverse Transcription Kit and TaqMan microRNA-specific primers (assay IDs: hsa-miR-16:000391, bkv-miRB1-5p:007796, bkv-miR-B1-3p:006801; Applied Biosystems). The qRT-PCR reaction contained 1 μL of reverse transcription product, 1x TaqMan Universal PCR mastermix, no AmpErase UNG, and 1 μL of primer mix. The complementary DNA (cDNA) was amplified using an ABI StepOnePlus real-time PCR system (Applied Biosystems). Reactions were incubated at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. A control sample was spiked with known concentrations of synthetic BKV microRNA mimic duplex oligonucleotides (bkv-miR-B1; IDT, Coralville, IA, USA). The sequences of the duplexes were 5’-AUCUGAGACUUGGGAAGAGCAU and 5’-UGCUUGAUCCAUGUCCAGAGUC [5 (link)].

Total RNAs (including miRNAs) derived from exosomes were extracted by a MirVana Paris Kit (Ambion, Austin, TX, USA) according to the manual. Denaturing solution (Ambion, Austin, TX, USA) was added to each sample. Total RNAs were dissolved in 100 μL RNase-free water. For further measurement, samples were stored in a −80°C refrigerator. The concentration of RNAs was assessed by a Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). After that, reverse transcription was applied to synthesize cDNA using the TaqMan Micro-RNA Reverse Transcription Kit (Thermo Fisher Scientific). The expression of ex-miRNAs was quantified by qRT-qPCR analysis according to the instructions of the manufacturer. We examined the obtained cDNA by qRT-PCR with TaqMan microRNA primers specific for miR-31, miR-192, and miR-375 (Thermo Fisher Scientific Inc., Waltham, MA, USA). A 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA) was employed for amplification and evaluation for miRNAs extracted from the exosomes. The miRNA expressions were calculated by 2^−ΔΔCt relative to RNU6B (U6): ΔC_t = C_tmiRNA − C_tU6.