Abi 7500ht instrument

SW1736 and KAT-18 cells in 96-well plates were allowed to reach approximately 70% confluence and then exposed to hypoxic conditions, or transfected with the indicated oligonucleotides before hypoxia exposure. The TRIzol reagent (Invitrogen) was used to isolate total RNA from tissue specimens and treated cells. cDNA synthesis was implemented using the High Capacity cDNA Synthesis kit (Applied Biosystems, Rotkreuz, Switzerland) for NEAT1 and TaqMan™ Advanced miRNA cDNA Synthesis kit (Applied Biosystems) for miR-206 and miR-599. qRT-PCR was performed on an ABI 7500HT instrument (Applied Biosystems) with SYBR Premix ExTaq mix (TaKaRa, Dalian, China). Relative expression of NEAT1, miR-206, and miR-599 were normalized according to the internal control of U6 snRNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated by the 2^−ΔΔCt method. Primers used for PCR were listed as: NEAT1 (Forward, 5′-TGGCTAGCTCAGGGCTTCAG-3′; Reverse, 5′-TCTCCTTGCCAAGCTTCCTT-3′); GAPDH (Forward, 5′-GAATGGGCAGCCGTTAGGAA-3′; Reverse, 5′-AAAAGCATCACCCGGAGGAG-3′); miR-206 (Forward, 5′-TGGAATGTAAGGAAGTG-3′; Reverse, 5′-CAGTGCGTGTCGTGGAGT-3′); miR-599 (Forward, 5′-GUUGUGUCAGUUUAUCAAAC-3′; Reverse, 5′-CTCCATATCGCACTTTAATCTCTAACT-3′); U6 snRNA (Forward, 5′-CTCGCTTCGGCAGCACA-3′; Reverse, 5′-AACGCTTCACGAATTTGCGT-3′).

Gene-specific primers were used in reverse transcription according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). qRT-PCR was performed on ABI 7500 HT Instrument (Applied Biosystems) using the Applied Biosystems 7500 Sequence Detection System. TaqMan (NoUmpErase UNG) Universal PCR Master Mix and specific primer and probe mix (Applied Biosystems) for each miRNA were used. PCR reactions were run in duplicates, and average threshold cycles and SD values were calculated.

Liver tissues were collected from the experimental pigs in the normal group (PN), FHF pigs in control group at day 3 after D-galactosamine induction (PC-D3), and pigs in hBMSCs transplantation group at week 3 and week 5 (PT-3W/PT-5W). To determine the effect of the transplanted hBMSCs on liver regeneration, liver tissues were analyzed using Hematoxylin and eosin (H&E) staining and immunohistochemistry with the human hepatocyte-specific marker ALB (Bethyl, Texas, USA) and an HSA (Abcam, Cambridge, UK). For H&E staining, the liver tissue section was heat-fixed at 60 °C for 1 hour and stained with H&E as described previously 9 (link).
Selected genes, including liver-specific genes and significantly differentially expressed miRNAs from the miRNA-seq experiments, were further validated by qRT-PCR using an ABI 7500HT instrument (Applied Biosystems, Waltham, MA, USA), as described previously 29 (link). The qRT-PCR was performed with a two-step protocol following the manufacturer's instructions. Briefly, total RNA was reverse transcribed with a miRNA-specific primer, and then qRT-PCR was performed with TaqMan probes (Invitrogen, CA, USA). The target genes were assayed in triplicate on each plate. Significantly differentially expressed miRNAs in the above cell and tissue samples were selected for qRT-PCR validation.