Mirneasy serum plasma spike in control cel mir 39

Total RNA from exosomes was isolated by using the miRNeasy Serum/Plasma Kit (QIAGEN). miRNeasy Serum/Plasma Spike-In Control (Cel-miR-39) (QIAGEN) was added to each exosome sample for normalization. Total RNA from cultured cells was isolated by using the Maxwell 16 LEV simply RNA purification kit (Promega, Madison, WI, USA).

TRIzol reagent (Invitrogen, USA) was used to extract total RNA from tissues, cells and CM derived-exosomes, which were purified using a miRNeasy Serum/Plasma Kit (Qiagen, Germany; Cat. Number: 217184), as instructed by the manufacturer. In addition, exosomal RNA was isolated directly from serum, using an exoRNeasy Serum/Plasma MidiKit (Qiagen, Hilden, Germany; Cat. Number: 77044). The miRNeasy Serum/Plasma Spike-In Control (cel-miR-39, Qiagen, Hilden, Germany; Cat. Number: 219610) was used as the serum miRNA expression profiling internal control, as instructed by the manufacturer. The cDNA of the RNAs were created with the aid of a PrimeScript™ RT Reagent Kit (TaKaRa, Japan; Code No. RR037A (miRNAs)/RR036A (mRNAs)). TB Green® Premix Ex Taq™ (TaKaRa, Japan, Code No. RR420A) was used to conduct the qRT -PCR, with the results recorded using ABI StepOne™ Software v2.3 (Applied Biosystems, USA). GAPDH functioned as an internal control for DYNLT1 mRNA levels and the relative miR-15b-3p expression of serum-exosomes were normalized to cel-miR-39, which was normalized to U6 in CM-exosomes, cells and tissues. The 2^−ΔCT formula was used to determine gene expression fold change. Additional file 7: Table S1 lists all primary sequences used.

Perforin was added to EVs (containing 6 μg protein) in Ca²⁺-HEPES buffer, according to the manufacturer’s instructions (Enzo Life Sciences Inc., New York, USA). The mixture was incubated at 37°C for 15 min. Next, the samples were divided in half, and RNase A (Qiagen, Hilden, Germany) was added to one of the two. After addition of RNase A, the samples were incubated at 37°C for 30 min. To isolate RNA, 700 μL QIAzol lysis reagent (Qiagen) was added to each sample, followed 5 min later by 10 μL of 10 nM miRNeasy Serum/Plasma Spike-In Control (cel-miR-39; Qiagen) (S4 Fig). Isolation of miRNAs from EVs was performed using a miRNeasy kit (Qiagen). Levels of miR-16 and cel-miR-39 were measured in TaqMan miRNA assays using real-time PCR. All TaqMan microRNA assays were purchased from Applied Biosystems (Massachusetts, USA). Real-time PCR was carried out using a StepOne Real-Time PCR System (Applied Biosystems), according to the manufacturer’s recommended program.