Quantifying miRNA-150-3p Expression

Total miRNA was extracted from tissues using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Real-time PCR assay was performed to detect the expression of miR-150-3p in tissues. Briefly, 10 µg of Micro RNA was subjected to reverse transcription. The cDNA was amplified through PCR and U6 was used as the endogenous control. The PCR primers used were as follows: miR-150 (GenBank accession number NR049590.1) forward, 5-CAG TAT TCT CTC CCA ACC CTT GTA-3 and reverse 5-AAT GGA TGA TCT CGT CAG TCT GTT-3; U6 (GenBank accession number XR003481952.1) forward, 5-ATT GGA ACG ATA CAG AGA AGA TT-3 and reverse, 5-GGA ACG CTT CAC GAA TTT G-3^{57 (link)}. The PCR conditions were: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 62 °C for 30 s, and 72 °C for 30 s^{57 (link)}. Real-time PCR was performed using SYBR Green PCR MasterMix (Applied Biosystems) on an ABI 7300HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). The expression of U6 was used to normalize the relative expression of miR-150-3p. For the purpose of data analysis, the comparative cycle threshold (CT) method was used.