Mirna assay kit

Mature miR-95-3p expression was detected using a miRNA-assay kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The U6 gene was used as a normalization control. All experiments were performed in triplicate and repeated once. To verify the integrity of HDGF expression, the GAPDH gene was used as an internal control. qRT-PCR was run using the following conditions: 30 cycles consisting of denaturation at 94 °C for 30 s, annealing at 56 °C (58 °C for GAPDH) for 30 s, and extension at 72 °C for 30 s. The primer sequences were as follows:miR-195 forward, 5′-ACACTCCAGCTGGGTTCAACGGGTATTTAT-3′ andreverse,5′-TGGTGTCGTGGA GGAGTCG-3'; U6 forward, 5′-CTC GCT TCG GCA GCA CA-3′ and reverse,5′-AACGCTTCA CGA ATT TGC GT-3'; HDGF forward, 5′-GAG GGT GAC GGT GAT AAG AA-3′ and reverse, 5′-GAAACATTGGTG GCTACA GG-3'; and GAPDH forward, 5′-TGCACC ACC AAC TGC TTA GC-3′ and reverse, 5′-GGC ATG CACTGTGGTCATGAG-3'. All samples were amplified in triplicate.