Omniscript rt

PBMCs were isolated from EDTA blood by density gradient centrifugation on Lymphoprep™ by Axis-Shield (Sentinel Diagnostic, Milan, Italy). Total RNA was extracted and purified using the Omega Bio-Tek E.Z.N.A.™ total RNA kit (VWR International, Milan, Italy) according to the manufacturer's instructions. After DNA digestion with DNase I enzyme (Qiagen, Milan, Italy), complementary DNA was synthesized from 1 μg of total RNA using Omniscript RT (Qiagen, Milan, Italy) and random hexamers.

Total RNA was extracted from H9c2 cells using Trizol reagent (Invitrogen) followed by DNase digestion for 10 min at room temperature with RNase-Free DNAse Set (Qiagen), and cleaned up with RNeasy Mini Kit (Qiagen). The quality of the RNA was determined by electrophoresis through agarose gels; only RNA samples with 28S:18S, rRNA ratio ≥ 2were used. Oligo-dT primer was used to target mRNAs present in the total RNA samples for conversion into cDNAs by reverse transcriptase (RT). Cleaned-up total RNA (2 μg) was reverse transcribed in a final volume of 20 μL containing 1x RT buffer, 0.5 mM dNTP Mix, 10 units of RNasin RNase inhibitor (Promega), 4 units of Omniscript RT (Qiagen), and 1 μM oligo-dT primer. Samples were incubated at 37°C for 60 min, followed by RT inactivation at 95°C for 5 min. Real-time PCR and gene-specific primers were used for quantification of TGF-β, PGC1-α, Nrf1, and YAP-responsive genes and FOXO3 responsive genes using Fast SYBR™ Green Master Mix (ThermoFisher, catalog number 4385614). The specificity of the reaction was verified by melt curve analysis. The relative quantification in gene expression was determined using the 2–ΔΔCt method (59 (link)). Using this method, we obtained the fold changes in gene expression normalized to internal control genes (β-actin, and/or GAPDH). The following primers were used for amplification

Total RNA from tissues and cells were isolated using TriZol. The first-strand complementary DNA was then synthesized using Omniscript RT (Qiagen) kit, and then used for quantitative RT–PCR using SYBR Green to quantify changes in mRNA. The data were analysed using the ΔΔC_T method and presented as arbitrary units16 (link)17 (link).

Right ventricles were crushed in TRIzol for RNA extraction as previously described 25. Cell RNA extractions were performed using the TaKaRa CellAmp Direct RNA kit (Clontech Saint‐Germain‐en‐Laye, France) as per the manufacturer's protocol. For miR‐322‐5p measurements, RNA was reverse‐transcribed (RT) using miR‐322‐5p and U6 Applied Biosciences TaqMan primers suspended in 1x TE buffer as per the manufacturer's instructions. cDNA was used for qPCR with TaqMan fluorescent probes for miR‐322‐5p and U6 according to the manufacturer's instructions. mRNA was measured via Omniscript RT (Qiagen, Manchester, UK) with random primers (Promega, Madison, WI, USA) as per the manufacturer's protocol. cDNA was diluted 1/10 with deionised water and used in qPCRs with QuantiFast SYBR Green (Qiagen) and specific primers (Sigma, Poole, Dorset, UK) for targets of interest as previously described 26.