Qiazol lysis buffer

60 mg pieces of frozen (− 80 °C) premotor cortex were homogenised (TissueRuptor II, Qiagen Ltd., Crawley, UK) on dry ice in QIAzol lysis buffer (Qiagen) and extracted using the RNeasy Lipid Tissue kit (Qiagen) with on-column DNase treatment (RNase-free DNase Set, Qiagen) according to the manufacturer’s instructions. RNA concentration was measured by Qubit™ RNA BR assay (Thermo Fisher Scientific Inc. Waltham, MA, USA) and RNA quality established by Agilent RNA 6000 Nano assay (Agilent Technologies, Inc. Santa Clara, CA, USA).

Distal colon tissues were collected in Magnalyser tubes (Roche, Basel, Switzerland) and ground in 700 µL of Qiazol lysis buffer (Qiagen, Hilden, Germany) with the FastPrep-24 5G (MPBio, Irvine, CA, USA). Total RNAs (including microRNAs) were purified using miRNeasy Mini Kit (Catalog number 217004, Qiagen) according to the manufacturer’s instructions.
RNA concentration and purity were determined using a UV spectrophotometer (NanoDrop ND1000, Thermo Fisher Scientific) by measuring the absorbance at 230, 260, and 280 nm. The integrity of the RNA was further checked with the Agilent 2100 Bioanalyzer, and RNAs with RIN values > 7 were used in the downstream RNAseq workflow.

For exosomal RNA sequencing, serum samples from the patients with 3 benign ovarian neoplasm and 5 EOC were used. Exosomes were isolated from serum using ExoQuick isolation agent (System Bioscience, Palo Alto, CA, USA) in accordance with the manufacturer’s instructions. Serum samples (1000 μL) were centrifuged at 3000× g for 15 min to remove cells and cell debris. The supernatants were mixed with ExoQuick reagent (System Biosciences, Palo Alto, CA, USA) and incubated for 30 min at 4 °C. After incubation, the samples were centrifuged at 1500× g for 30 min to generate an exosome pellet that was resuspended in 100 μL of sterile phosphate-buffered saline (PBS). Total RNA was extracted from the exosomes using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Exosome suspensions were mixed with 700 μL QIAzol lysis buffer (Qiagen), and the mixtures were processed according to the manufacturer protocol. The RNA was eluted in 20 μL RNase-free water (Qiagen). The size distribution of purified RNA was assessed using an Agilent 2100 Bioanalyzer with an RNA Pico Chip and Small RNA Chip (Agilent Technologies, Santa Clara, CA, USA).

For in vitro free uptake experiments, the total RNA was extracted using the NucleoSpin RNA XS (Macherey Nagel, Hoerdt, France) according to the manufacturer’s recommendations. For transfection experiments the SuperScript™ IV CellsDirect™ cDNA Synthesis kit (Thermo Fisher Scientific) was used according to manufacturer’s recommendations. For in vivo experiments, mouse organs were crushed in 2 mL tubes pre-filled with ceramic mixture in Precellys^®Cryolys^® Evolution (Bertin, Montigny le Bretonneux, France) with a QIAZOL lysis buffer (Qiagen, Venlo, The Netherlands). A volume of 150 µL of chloroform (Sigma Aldrich) was added to 750 µL of tissue homogenate and a phenol/chloroform separation was performed using centrifugation for 15 min at 6000× g at 4 °C. Aqueous phases were recovered and RNA extraction was performed with the RNeasy 96 QIAcube HT Kit (Qiagen) according to the manufacturer’s recommendations, in a QIACUBE HT instrument (Qiagen). Quality and quantity of total RNA was determined with DNF-471 RNA Kit-15 nt (Agilent, Santa Clara, CA, USA) in a Fragment Analyzer 5300 (Agilent). For reverse transcription (RT), 500 ng of total RNA were used (except for cells treated with SuperScript™ IV CellsDirect™ cDNA Synthesis), and cDNA synthesis was performed using the High-Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

Adult mouse tissues were freshly collected from one male and one female 8-week-old C57BL/6 J mice. Tissues were immediately homogenized using IKA T10 basic ULTRA-TURRAX homogenizer in an appropriate volume of QIAzol lysis buffer (Qiagen). Total RNA was extracted from cell cultures and from murine tissues by miRNeasy mini kit (Qiagen). MicroRNA-125a was quantified along with RNU6B (reference transcript) by RT-qPCR with TaqMan® miRNA assays from Applied Biosystems according to the manufacturer’s protocol. For quantification of SPACA6 and TNFAIP3 transcripts, total RNA was retrotranscribed by Transcriptor High Fidelity cDNA Synthesis Sample kit (Roche) using random examer primers. Then standard SYBR Green Real-time qPCR assays were performed with the following primers: SPACA6 transcription variant 2, 5′-GGGGAGAGGATGGAGAGC-3′ and 5′-TCATTTTCTCCGCAGCATC-3′^{45 (link)}; TNFAIP3, 5′-TCAACTGGTGTCGAGAAGTCC-3′ and 5′-CCAAGTCTGTGTCCTGAACG-3′^{46 (link)}; GAPDH (reference gene) 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTT-3′. Finally, the expression levels of miR-125a, SPACA6 and TNFAIP3 were normalized to their respective reference genes by using the 2^−ΔCt method and reported as arbitrary units (AU). Comparison of data sets was performed by Student’s t-test and a value of p < 0.05 was considered significant.