Total RNAs in tissues and cells were isolated using the TRIzol method (Invitrogen) according to the manufacturer’s instructions. In the process of serum RNA extraction, 1 ug lyophilized cel-miR-39-3p (Qiagen, Hilden, Germany) was added as exogenous reference according to the provider recommendations and a miRNeasy serum/plasma kit was used to extract serum cell-free miRNAs according to manufacturer’s protocol. The Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, NC, USA) was used to detect RNA concentration. The expression levels of miRNAs were determined using All-in-One miRNA qRT-PCR Detection Kit (GeneCopeia, Rockville, MD, USA) in the ABI 7500 System (Applied Biosystems, Foster City, CA, USA) according to the protocol. The relative expression of miR-371a-5p in tissues and cells were normalized to U6 and that in serum to cel-miR-39 using the 2−ΔΔCt method. The primers were as follows:
cel-miR-39, 5′-CAGAGTCACCGGGTGTAAAT-3′;
miR-371a-5p, 5′-TGCGGACTCAAACTGTGGGGGC-3′;
u6, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′.
cel-miR-39, 5′-CAGAGTCACCGGGTGTAAAT-3′;
miR-371a-5p, 5′-TGCGGACTCAAACTGTGGGGGC-3′;
u6, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′.
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