Qscript microrna cdna synthesis kit

RNA was isolated using Qiazole/Trizole as per the manufacturer's instructions. MicroRNAs were converted to cDNA using the qScriptTM microRNA cDNA synthesis kit (Quanta Biosciences). Real-time qPCR was performed using Brilliant II SYBR Green RT–qPCR kit. MiR-29b, actin (beta), Rnu6 or Snord47 was used as internal controls. qPCR with reverse transcription (RT–qPCR) of Sirt1, Trpm3 and Gapdh were performed using the Brilliant II SYBR Green RT–qPCR kit. Gapdh was used as an internal control. Primer sequences are provided in Supplementary Table 1.

RNA was isolated using Qiazol/Trizol, as per the manufacturer's instructions. miRs were converted to cDNA using the qscriptTM microRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA). RT‐qPCR for miR‐204 (mature and precursor), nppa, nppb, β‐mhc, Col1a1, Col1a2, Col3a1, Sirt1, Apln, Apela and Aplnr was performed using the Brilliant II SYBR Green RT‐qPCR kit. 18S was used as an internal control. The primer sequences are provided in Table S3.

RNA from total pig plasma (n = 6) and plasma-derived EV (n = 4) was extracted using the miRNeasy kit for plasma (Qiagen) and Trizol LS, respectively. Both protocols were performed according to the manufacturer’s descriptions. To correct for isolation variability and to enable comparative analysis of total plasma and plasma EV, C. Elegans miRNA-39 (Quanta Biosciences) was added to the lysis buffer equalized to the starting amount of plasma. RNA quantity and quality were measured with the Nanodrop (NanoDrop Products) and the 2100 Small RNA Assay Bioanalyzer (Agilent). cDNA was synthesized with qScript™ microRNA cDNA Synthesis Kit (Quanta BioSciences), following the manufacturer’s protocol. Quantitative RT-PCR (qRT-PCR) was performed in 12.5 μl duplicate reactions with PerfeCTa SYBR Green SuperMix (BioSciences), the PerfeCTa Universal PCR Primer (Quanta Biosciences), and primers specific for miRNA-1, miRNA-21, miRNA-133b, miRNA-146a, miRNA-208b, and miRNA-499a.
The cycle number that exceeds the fluorescence threshold is the threshold cycle (Ct value). Ct values that exceeded 40 cycles were treated as Ct 42. At missing time points, the average of the other pigs in that specific group was used as the cycle number for that time point. Ct values were normalized by using the average Ct value of the spike-in miRNA.

Venous blood was collected and kept on ice for 45 min. After centrifugation at 2000×g and 4 °C and for 10 min, the supernatant serum was collected and stored in aliquots at − 80 °C until further analysis. Samples with visible haemolysis were excluded. Total small RNAs were extracted from 200 μL serum using the miRNeasy serum plasma isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Synthetic C. elegans (C) miR-39-3p (Qiagen, Hilden, Germany) was spiked-in at a final concentration of 1.6 × 108 copies/μL after the initial denaturation, prior to extraction, to correct the extraction efficiency. The total RNA was eluted in 14 μL of RNase-free water. A fixed volume of 7 μL of eluate was used as input for the cDNA synthesis. RNA was converted to cDNA using the qScript™ microRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). qPCR was performed with PerfeCta® SYBR® Green Supermix on an Applied Biosystems (Foster City, CA) 7900 Real Time PCR System with the following conditions: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 15 s and 70 °C for 15 s. MicroRNA expression levels were normalized to the mean of spiked-in miR C-miR-39 and presented as 2^−ΔCt values.