TaqMan microRNA Assays (Applied Biosystems) was used for detection of miR-34a-3p and miR-34a-5p expression according to manufacturer’s protocol. Briefly, reverse transcription (30 min, 16 °C; 30 min, 42 °C; 5 min 85 °C) was performed using a TaqMan stem loop primer, 15 ng RNA, TaqMan primers and miR reverse transcription kit (Applied Biosystems). qRT- PCR was performed using the TaqMan Universal PCR Master Mix according to the manufacturer’s protocol. RNU-48 was used to normalize the Ct values between the samples. In experiments involving miR analysis of exosomes or plasma, synthetic C. elegans (cel)-miR-39 was spiked during the total RNA isolation process and used as normalizer. All experiments were performed in triplicate. miR levels were normalized and the relative expression levels of specific miR were presented by 2–ΔΔCt.
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