Qiazol kit

Total RNA was isolated from cells and tissue using the Qiazol kit (Qiagen), as per manufacturer instructions, and kept frozen at −80°C until extraction. Total mRNA was extracted using the RNeasy kit (Qiagen), according to manufacturer protocols. cDNA synthesis was performed on 1 μg total RNA using the iScript reverse transcriptase kit (Biorad). Quantitative PCR was performed using commercially available mouse primers for Gapdh, Arg1, Il10, Tgfb, Ccl2, Il1b, Il12b, Cxcl9, and Ifna using Quantitect primer assays (Qiagen). SYBR Green 2X Master Mix was used for qPCR analysis (Biorad). mRNA levels were normalized to Gapdh levels (dCt = Ct target gene – Ct Gapdh) and reported as relative mRNA expression [2^(−ddCt) where ddCt = dCt experimental – dCt control] (Qiagen).

RNA was extracted from cells or tissues using a QIAzol Kit (Qiagen, Hilden, Germany). cDNA was synthesized using a commercially available kit (Invitrogen Corp.). Total RNA was incubated with random hexamers and a dNTP mix at 70 °C for 10 min. After incubation, M-MLV, RNase inhibitor, 0.1 mmol/L DTT, and 5 × FS buffer were added according to the manufacturer’s instructions. The samples were then incubated at 42 °C for 1 h. The cDNA synthesis was terminated at 95 °C for 5 min. Reaction mixture aliquots were used as templates for PCR. Amplification reactions were performed using a DNA thermal cycler (MasterCycler; Eppendorf, Hamburg, Germany). Quantitative real-time PCR analysis was performed in triplicate with a Rotor-Gene Q (Qiagen) using TOPreal™ qPCR 2 × PreMIX (Enzynomics, Daejeon, Korea). The amounts of mRNAs were normalized to the 18 S rRNA endogenous control. Oligo sets for Nppa and Nppb were purchased (Bioneer, Daejeon, Korea). The specific sequences for PCR were as follows:
18S rRNA, sense: 5ʹ-GTAACCCGTTGAACCCCATT-3ʹ.
18S rRNA, antisense: 5ʹ-CCATCCAATCGGTAGTAGCG-3ʹ.
PPP2CA, sense: 5ʹ-GATCACACAAGTTTATGGTTTCTATGATGAA-3ʹ.
PPP2CA, antisense: 5ʹ-TATGATCCAGTGTATCTATAGATGGCGAG-3ʹ.

Prefiltered plasma was mixed with 2x binding buffer (XBP) at a ratio of 1:1 and then added to the exoEasy membrane affinity column. After centrifugation, the effluent was discarded, and washing buffer (XWP) was added to the column. After centrifugation, the flow through was discarded. QIAzol kit (QIAGEN, Hilden, Germany) was used to lyse the vesicles. Following centrifugation, the lysate was collected and chloroform was added and then thoroughly mixed. After centrifugation, ethanol was added to the aqueous phase. The sample–ethanol mixture was added onto RNeasy MinElute spin columns. After centrifugation, columns were washed once with buffer RWT, then twice with RPE buffer, before eluting RNA in water (15 (link)). The total RNA extracted from cirexos was subjected to high-throughput sequencing.