RT–qPCR of exosomal RNA expression levels

The expression of IFN-γ mRNA in sEVs was detected by RT–qPCR according to the manufacturer’s instructions. Briefly, total RNA was isolated from sEVs by using TRIzol (Invitrogen) and was reverse-transcribed into cDNA with a Reverse Transcription Kit (Thermo Fisher Scientific). Gene expression was measured by using the SYBR Green qPCR kit (BioRad). Expression values were normalized to that of U6. Gene-specific primers included U6 forward (5’-CTCGCTTCGGCAGCACA-3’), U6 reverse (5’-AACGCTTCACGAATTTGCGT-3’), IFNG (human) forward (5’-ACAGCAAGGCGAAAAAGGATG-3’), IFNG (human) reverse (5’-TGGTGGACCACTCGGATGA-3’), Ifng (mouse) forward (5’-CAGCAACAGCAAGGCGAAAAAGG-3’), and Ifng (mouse) reverse (5’-TTTCCGCTTCCTGAGGCTGGAT-3’).

The absolute copy number of target mRNA in sEVs was also determined by qPCR results. The average number of target mRNAs per sEV was calculated by dividing by the sEV number measured using NS300. Briefly, the isolated RNA was first reverse-transcribed into complementary DNA (cDNA) by using the TaqMan^TM reverse transcription kit (Life Technologies, Carlsbad, CA), following the manufacturer’s protocol. The subsequent quantitative polymerase chain reaction (qPCR) analysis was done in triplicate with 100 ng of DNA in a 20 μL reaction volume. Each 20 μL reaction contained 10 μL of TaqManTM Fast Advanced Master Mix, 1 μL of the Gene copy number assay (TaqMan^TM Ifng Gene copy number assay Mm00734344_cn), and 9 μL of the DNA template. The qPCR conditions included an initial denaturation step at 50 °C for 2 min, followed by a 10 min step at 95 °C. Subsequently, a total of 40 cycles were performed, consisting of denaturation at 95 °C for 15 s, followed by annealing and extension at 60 °C for 1 min.