Mircury rna isolation kit cell plant

EV RNA extraction was performed using the miRCURY RNA Isolation Kit - Cell & Plant (#300110, Exiqon) following the manufacturer’s instructions. In brief, exosome pellet was lysed with the provided lysis solution supplemented with 96–100% ethanol, and the mixture was loaded to the column. The EV RNA was washed and eluted with 15–50 µL of RNase-free water. The extracted RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). RNA size was confirmed using Agilent RNA 6000 Pico Kit and Small RNA Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).

miRNAs contained in sEVs from aPL, PAPS and HD (n = 8) were extracted following the instructions of miRCURY RNA Isolation Kit—Cell & Plant (Exiqon, Woburn, MA, USA). miRNA samples were processed and sequenced (single end, 50 nts, 1 × 50, v4) by CNAG (Barcelona, Spain) on a HiSeq2500 Illumina device with a read depth of >10 M reads/sample. Subsequent quality control, data processing and analysis were performed as previously described [48 (link),49 (link),50 (link)]. Data were deposited in the Gene Expression Omnibus (NCBI) with the number GSE220791 (more details in Supplementary Materials).

Total RNA was isolated from cell lines and exosomes using the miRCURY RNA Isolation Kit – Cell & Plant (Exiqon, Woburn, MA) followed with spectrophotometry (NanoDrop, Thermo Scientific) for quantification and qualitative analysis. Equal amounts of total RNA was reverse-transcribed by oligo (dT) priming using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer's instructions. Real-time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) and the SYBR Green PCR Master Mix (Applied Biosystems) as described previously [40] (link). Glut1 and β-Actin primers were purchased from SABiosciences (Frederick, MD).

RNA from urinary pellets or exosomes were extracted using the miRCURY™ RNA Isolation Kit-Cell & Plant (Exiqon, Woburn, MA, USA, more details in SI), following the manufacturer’s instructions. Quantification and evaluation of the RNA quality was assessed by Bioanalyzer PicoChip analysis.

To extract RNA from uEVs isolated by ultracentrifugation, we employed miRCURY™ RNA Isolation Kit Cell & Plant (Exiqon). In average, 1.5e⁷ vesicles were used per retrotranscription reaction. In addition, a set of samples was extracted by Norgen Biotek Exosomal RNA purification kit, following the manufacturers' instructions. For cell lines, RNA was extracted using NucleoSpin^® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). cDNA was synthesized from 0.1–1 μg of RNA using Superscript III (Life Technologies) following the manufacturer's recommendations. For prostate tissue samples, RNA was extracted as reported in [16 ]. Quantitative Real Time PCR (Taqman qRTPCR) was performed as previously described [18 (link)]. Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table S2. β-ACTIN (Hs99999903_m1) and GAPDH (Hs02758991_g1) housekeeping assays were from Applied Biosystems and showed similar results.

EVs were isolated from 1 mL of supernatant of centrifuged pre‐resection pleural lavage fluid using miRCURY Exosome Isolation Kit‐Cells, urine, and CSF (Exiqon) according to the manufacturer's instructions. This kit is a viable alternative to ultracentrifugation, even with limited biological samples.25 Representative isolated EV samples were verified using nanoparticle tracking analysis by Theoria Science. Positive and negative surface protein markers of exosomes were confirmed using the Exo‐Check Exosome Antibody Arrays (System Biosciences) according to the manufacturer's instructions.
Total RNA was isolated from 100‐µL EV samples using the miRCURY RNA Isolation Kit‐Cell&Plant (Exiqon), following the manufacturer's instructions. Reverse transcription reaction was performed with 5 µL of total RNA using the TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays RT primer (Thermo Fisher Scientific).