Quantitative RT-PCR confirmation of temperature effects on miRNA expression pattern in human SAECs. Human SAECs from two lots were incubated for 24 h at 32°C, 37°C, or 39.5°C without or with 1 ng/mL rhTNFα. Total RNA was collected and levels (A) hsa-miR-92a-1-5p, (B) hsa-miR-27b-5p, (C) hsa-miR-1260a, (D) hsa-miR-27a-5p, (E) hsa-miR-181a-3p, and (F) hsa-miR-18b were analyzed by qRT-PCR as described in Materials and Methods and expressed as a fold-change versus 37°C cells without TNFα. The right-hand axis displays the total expression level in copies per cell, which was calculated as described in Materials and Methods. Mean ± SEM. (*) P < 0.05 versus 37°C; (†) P < 0.05 versus 39.5°C; (§) P < 0.05 versus 37°C with TNFα; (¶) P < 0.05 versus 39.5°C with TNFα, n = 4. The temperature-responsive miRNAs were cloned into T-Vector and sequenced (inset).
