Quantification of Exosome-associated Genes

The indicated numbers of LUHMES cells, ACs, and MG were seeded in a plate dish. The next day, 1 or 10 pg/mL IL-6 was added and cells were collected 24 h later. Total RNA including miRNA was purified with an miRNeasy Kit (217084; Qiagen, Venlo, the Netherlands). qRT-PCR assay was then performed as described previously [22 (link)], for which the primer sets were as follows: Hs00233521_m1 for CD9, Hs01041238_g1 for CD63, Hs01002167_m1 for CD81, Hs01075664_m1 for IL-6R, and Hs01060665_g1 for β-actin (Applied Biosystems, Foster City, CA, USA). Values were normalized to those of human β-actin. The comparative cycle time method was used to quantify gene expression. All samples were analyzed in duplicate in each experiment.