Nucleospin mirna

RNA was extracted from HeLa cells using the RNeasy Mini kit (Qiagen, Hilden, Germany), and cDNA was generated using PrimeScript reverse transcriptase (Takara). Small RNA was extracted from RIP assay using NucleoSpin miRNA (Takara), and cDNA was generated using miRCURY LNA™ Universal RT microRNA PCR Universal cDNA synthesis KitII (EXIQON, Copenhagen, Denmark). PCR was performed using the SYBR® Premix Ex Taq™ II (Takara) (for mRNA) or miRCURY LNA™ Universal RT microRNA PCR ExiLENT SYBR® Green master mix (EXIQON) (for small RNA). The relative RNA expression levels were normalized to GAPDH or miRCURY LNA UniRT PCR Control primer mix UniSp6 (203954, EXIQON). The sequences of primers used to amplify each gene were: 5′-AGGTGGAGGAGTGGGTGTCGCTGTT-3′ and 5′-CCGGGAAACTGTGGCGTGATGG-3′ (GAPDH); 5′-CAGCATCCACAGTGCAGATC-3′and 5′-GCACCTCAGAGAAGCAATGC-3′(NOP56); 5′-TGGCAGCTATGTGTCTTGGA-3′and 5′-TGCCAGCCATACCATTCTCT-3′(NOP58); 5′-TGCTCTGATGAAATCACTAA-3′and 5′-AATCAGACAGGAGTAGTCTT-3′(SNORD49A); miRCURY LNA UniRT PCR Reference primer mix SNORD44 (203902, EXIQON).

Total RNAs of hBSMCs and mouse BSM tissues were extracted using NucleoSpin™ miRNA (TaKaRa Bio, Inc., Shiga, Japan) according to the manufacturer’s instruction. cDNAs were prepared from the total RNA by using PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer’s instructions. cDNA samples were subjected to PCR with Quick Taq™ HS DyeMix (TOYOBO Co., Ltd., Osaka, Japan) in a final volume of 10 µL. The PCR primer sets used are shown in Table 1 (for human) and Table 2 (for mouse), which was designed from published database, BLAST. The thermal cycle profile used was (1) denaturing for 30 s at 94 °C, (2) annealing primers for 30 s at 60 °C, (3) extending the primers for 1 min at 68 °C, and the reaction was run for 40 cycles. The PCR products were subjected to electrophoresis on 2% agarose gel and visualized by ethidium bromide staining.