Seramir

miR-214 and CaMKII mRNA levels were determined by using quantitative RT-PCR as previously reported [36 (link), 37 (link)]. Briefly, total RNA was extracted from the exosomes by SeraMir (System Biosciences) following the manufacturer's instructions, and cell RNA was extracted by an RNAprep pure Cell/Bacteria kit (Tiangen, Beijing, China). miR-214 levels were quantified with a stem-loop real-time PCR miR kit (RiboBio, China). The miR primer was also purchased from RiboBio (China). The purity of the isolated RNA was determined by the OD260/280 ratio using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). Isolated RNAs were reverse transcribed using a PrimeScript RT Reagent kit (TaKaRa, Kusatsu, Shiga, Japan). cDNA was used for quantitative PCR on a Bio-Rad Real-Time PCR system (Bio-Rad, Hercules, CA, USA) using a SYBR kit (Bio-Rad, USA). Amplification was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, and 55.7°C for 30 s. The difference in the expression levels between the treatments was then calculated using the following equation: relative gene expression = 2 − (ΔCt, sample − ΔCt, control). U6 and β-actin were used as internal controls for miR-21 and CaMKII mRNA quantitation, respectively.

Total RNA was extracted from exosomes by SeraMir (System Biosciences) following the manufacturer's instructions and cellular RNA was extracted using the RNAprep pure cell/bacteria kit (TIANGEN, Beijing, China). The purity of the isolated RNA was determined at OD260/280 using a Nanodrop ND-1000 (Thermo Scientific), and integrity was assessed by 1% agarose gel electrophoresis. RNAs were reverse-transcribed to first-strand cDNA using the PrimeScipt RT Reagent Kit (TaKaRa, Dalian, China). The cDNA was subjected to qRT-PCR using the SYBR kit (TaKaRa, Dalian, China). miR-128-3p expression was quantified with a stem-loop real-time PCR miRNA kit (Ribobio, Guangzhou, China). The threshold cycle (Ct) was determined for each reaction using the 2^−ΔΔCt method, and the expression of each gene of interest was normalized to that of the endogenous control gene (U6 for miRNAs or GAPDH for mRNAs). The primer sequences in the study are shown in Supplementary Table 1.