Exorneasy serum plasma midi kit

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)].

At each visit, 2.5 mL of blood and 50 mL of urine were collected. After collection, the blood was stored in PAXgene Blood RNA tubes, and total RNA was extracted using a PAXgene Blood RNA Kit (PreAnalytiX; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The urine was centrifuged at 2000× g for 20 min, the pellet was transferred and stored at −70 °C until use. Total RNA was extracted from the urinary pellets using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Urinary exosomal RNA was isolated from 1 mL of urinary supernatant using spin column-based exoRNeasy serum/plasma midi kits (QIAGEN GmbH, Hilden, Germany). Then, 20 μg of urinary exosomal proteins was separated by SDS-PAGE, transferred to a nitrocellulose membrane, probed with the appropriate primary antibodies, and incubated with horseradish peroxidase-linked secondary antibodies. Blots were visualized with enhanced chemiluminescence detection reagents and quantified using ECL hyperfilm. Band volumes were measured by densitometry in at least three different experiments. Primary antibodies against the following proteins were used: tetraspanin-1 (H00010103, 1:500; Abnova, Taipei, Taiwan) and hemopexin (ab124935, 1:500; Abcam, Cambridge, UK). The details are shown in our previous studies [7 (link),10 (link),11 (link),12 (link),13 (link)].

RNA from CTC samples was extracted with Total RNA Purification Micro Kit (Norgen Biotek, Thorold, ON, Canada) and eluted in 30 µL elution buffer. RNA from plasma was extracted with QIAamp circulating nucleic acid kit (QIAGEN, Hilden, Germany) and eluted in 65 µL elution buffer. Samples processed with this kit are referred to as ctRNA even for healthy controls that would only have normal cell free RNA. RNA from exosomes was extracted with Qiagen exoRNeasy serum/plasma Midi kit (QIAGEN, Hilden, Germany) and eluted in 30 µL. A total of 15 µL eluted RNA was reverse transcribed using SensiFAST cDNA synthesis kit (Bioline, Alexandria, Australia).

Exosomal RNA from early pregnancy plasma (n = 3) and non-pregnancy plasma (n = 3) was isolated by an exoRNeasy Serum/Plasma Midi Kit (#77044, Qiagen, Germany) according to the manufacturer’s instructions. The integrity, purity, and concentration of RNA were determined using a RNA Nano 6000 Assay Kit with an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), NanoPhotometer® spectrophotometer (IMPLEN, USA), and Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, USA), respectively. Then, sequencing was performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).