Sybr green kit

Total RNA was isolated by using NucleoZOL reagent (MACHEREY-NAGEL GmbH & Co.KG., Duren, Germany) in accordance with the manufacturer’s instructions. RNA concentration was measured using a spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). cDNA was generated with 1 μg of total RNA using a Reverse Transcription System kit (Promega, Fitchburg, WI, USA). PCR was performed using a SYBR Green kit (Agilent Technologies, West Cedar Creek, TX, USA) and primers specific to the target genes, namely: PKC-α: 5′-CCCATTCCAGAAGGAGATGA-3′ (forward, accession no. NM_011101.3) and 5′-TTCCTGTCAGCAAGCATCAC-3′ (reverse, accession no. NM_011101.3); α-SMA: 5′-ACTGCCGAGCGTGAGATTGT-3′ (forward, accession no. XM_006526606.2) and 5′-TGATGCTGTTATAGGTGGTTTCG-3′ (reverse, accession no. XM_006526606.2); TGF-β1: 5′-CGAAGCGGACTACTATGCTAAAGAG-3′ (forward, accession no. NM_011577.2) and 5′-TGGTTTTCTCATAGATGGCGTTG-3′ (reverse, accession no. NM_011577.2); GAPDH: 5′-CAGCCTCGTCCCGTAGACA-3′ (forward, accession no. XM_017321385.1) and 5′-CGCTCCTGGAAGATGGTGAT-3′ (reverse, accession no. XM_017321385.1). Gene expression was evaluated by the ΔΔCT method with GAPDH as a housekeeping gene.

Results of high-throughput analyses (small-RNA sequencing or PCR arrays) were validated by RT-qPCR on the same plasma samples used for sequencing (n = 8 heifers/group). cDNA was generated as described above and diluted for use in 10 μL qPCR reactions using Qiagen SYBR Green kits in an Agilent Mx3005P qPCR system (Agilent Technologies, USA). Raw fluorescence data were processed using Agilent MxPro software. A fluorescence threshold of 0.1 was set for all experiments. The amplification efficiency generally ranged between 85 % - 115 %, with R² > 0.85. Cq-values and gene expression data were processed using Microsoft Excel and statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, USA). Specifically, data were log2 transformed and tested for normality as described for the small-RNA sequencing data, and Dunn’s multiple comparison tests (non-parametric) were used to generate P values for the comparisons of miRNA levels between non-pregnant (NP) and pregnant groups (P16 or P24).

cDNA was generated as described above and diluted for use in 10 μL qPCR reactions using Qiagen SYBR Green kits in an Agilent Mx3005P qPCR system (Agilent Technologies, USA). Raw fluorescence data were processed using Agilent MxPro software. A fluorescence threshold of 0.1 was used to determine Cq values for all experiments. The amplification efficiency ranged between 88% and 109%, with R2 > 0.85. Data were processed using Microsoft Excel (Microsoft Corporation). Statistical analyses were performed in GraphPad Prism 6 (GraphPad Software, USA) using repeated-measures ANOVA followed by Dunnett’s tests or 2-sample t-tests if comparisons involved only two means. Statistical significance was set to P < 0.05. For selected circulating miRNAs, normalised expression levels were correlated with plasma progesterone levels using Spearman’s correlation (ρ) in GraphPad Prism 6 (GraphPad Software).