To verify miRNA sequencing results, miRNA expression was assayed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using the Taqman MiRNA Reverse Transcript Kit (Cat# 4366596, Thermo Fisher, Waltham, MA, USA). SYBR Green-based qRT-PCR was used to measure the relative expression of selected target genes. Five hundred nanograms of the total RNA were reverse transcribed to generate complementary DNA (cDNA) employing the High Capacity cDNA Reverse Transcript Kit (Thermo Scientific, Rockford, IL, USA). The QuantStudio™ 3 Real-Time PCR System (ABI) was used for these assays. Relative expression was calculated using the 2−ΔΔCT method [23 (link)].
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