Prism 7900 ht sequence detector

Total RNA was isolated using the miRNeasy Mini kit (Qiagen) with the optional DNase I digestion step. The TaqMan^® MicroRNA Reverse Transcription Kit (ThermoFisher) was used for first strand synthesis and TaqMan^® MicroRNA assay kits were used for quantitative analysis of microRNAs. We utilised a PRISM 7900 HT Sequence Detector from Applied Biosystems to conduct real‐time TaqMan^®Assays, employing the TaqMan^® Universal PCR Master Mix from Invitrogen. To determine the relative levels of the miRNAs, we employed the comparative ΔCt method,⁵⁹ which involved normalising the data to RNU48 gene expression. Each miRNA was measured in three separate experiments, with three biological replicas in each experiment, each assayed in triplicate. The probes assay kits used for miRNA detection are listed in Table S1.

After iPIC differentiation, cDNA samples were synthesized from lysates using TaqMan Gene Expression Cells-to-Ct kits (Thermo Fisher Scientific) according to the manufacturer's instructions, followed by quantitative real-time polymerase chain reaction analysis using a Prism 7900HT sequence detector (Thermo Fisher Scientific). The thermal cycling parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The mRNA levels were analyzed with the comparative Ct method (2-ΔΔCt) using RPLP0 as the housekeeping gene. The TaqMan Gene Expression assays (Thermo Fisher Scientific) used herein were as follows: Hs00170586_m1 (FGB), Hs00356521_m1 (AGR2), and Hs00420895_gH (RPLP0).

Neonatal rat cardiomyocytes were seeded in a collagen I-coated 96-well plate, then cultured and maintained for a week before the ligand stimulation assay. The culture medium was replaced by HBSS buffer containing 20 mM HEPES and then stimulated with the indicated ligands for 15 to 90 min. After stimulation, cDNA samples were synthesized from lysates using TaqMan Gene Expression Cells-to-Ct kits (Thermo Fisher Scientific) according to the manufacturer’s instructions, followed by quantitative real-time polymerase chain reaction analysis using a Prism 7900HT sequence detector (Thermo Fisher Scientific). Thermal cycling parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. The mRNA levels were analysed with the comparative Ct method (2^−ΔΔCt) using Rplp0 as the housekeeping gene. The TaqMan Gene Expression assays (Thermo Fisher Scientific) used herein were as follows: Rn01533237_m1 (Nr4a1), Rn00570936_m1 (Nr4a2), Rn01354012_m1 (Nr4a3), Rn02396759_m1 (c-fos), and Rn03302271_gH (Rplp0).