Mir 16

Taqman miRNA assay for miR-10b, miR-16, and miR-21 were purchased from ThermoFisher Scientifics.

Serum miR-150 level was quantified in triplicate by qRT-PCR using TaqMan MicroRNA Assay Kits (Applied Biosystems, Foster City, CA). The reverse transcription reaction was performed in a 20 μl reaction volume using specific primer for miR-150 contained in the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). For synthesis of cDNA, the reaction mixtures were sequentially incubated at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. According to the standard TaqMan MicroRNA assay protocol, real-time PCR was performed in ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) with the following cycle: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Each PCR mixture (20 μl) included the reverse transcription products, TaqMan 2X Universal PCR Master Mix without UNG Amperase, miRNA-specific TaqMan probes, and primers supplied by Applied Biosystems. The cyclethreshold (Ct) values were calculated with the SDS 2.0.1 software (Applied Biosystems, Foster City, CA). The formula 2^−⊿Ct was used to calculate the miRNA levels in serum, where ⊿Ct = mean (Ct of internal references) − Ct of target miRNA. The relative expression levels of miR-150 were calculated and normalized to miR-16 (Applied Biosystems, Foster City, CA) using the comparative⊿Ct method and the equation 2 ^−⊿Ct, as described previously [23 (link)].

A TaqMan miRNA assay was used to detect the contents of miRNAs. The single-stranded cDNA was synthesized using the TaqMan miRNA reverse transcription kit (Applied Biosystems; Foster City, CA, USA) and then amplified by using the TaqMan universal PCR master mix (Applied Biosystems; Foster City, CA, USA) together with the following miRNA-specific TaqMan MGB probes: Let-7b, miR-125a, miR-181b, miR-16, miR-30b, and miR-101 (Applied Biosystems; Foster City, CA, USA).

Following serum collection, total RNA, including miRNAs, was extracted from serum samples using the mirVana PARIS Kit (Applied Biosystems), according to the manufacturer’s instructions. A total of 10 ng of the extracted RNA was subjected to Reverse Transcriptase PCR using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions. Real-Time PCR amplification was performed using TaqMan MicroRNA Assays to measure miRNAs levels. miRNA detection assays specific for miR-1, miR-133a, miR-133b and miR-206 (Applied Biosystems) were carried out according to the manufacturer’s instructions. miRNA expression was normalized to the miR-16 (Applied Biosystems). Data analysis was performed using the SDS 2.4 Real-Time PCR data analysis software (Applied Biosystems).