RT-qPCR validation

RT-qPCR was performed to validate the expression level of 9 (random selected) out of 22 annotated DEmiRNAs. For cDNA synthesis, 1 μg of total RNA from each sample was used as input material (miScript II RT kit, Qiagen, USA). Following manufacturer’s instruction, 4 μL of 5 × miScript HiSpec Buffer, 2 μL of 10 × nucleotide triphosphate mix, 2 μL miScript reverse transcriptase mix, and 10 μL of RNAse free water, were mixed to reach a total volume of 20 μL. Reactions were incubated first for 60 min at 37 °C followed by 5 min at 95 °C to inactivate the reverse transcriptase.

qPCR (miScript PCR system, Qiagen, USA) reactions were set up in a total volume of 25 μL containing 12.5 μL 2 × QuantiTec SYBR Green PCR Master Mix, 2.5 μL 10 × miScript Universal Primer, 2.5 μL miRNA forward primer (5 mmol/L), 2.5 μL template cDNA (diluted ten times in RNAse free water) and 5 μL of RNAse free water. The polymerase was activated during 15 min at 95 °C followed by a 3-step amplification program as follows: denaturation 15 s 94 °C, annealing 30 s 57 °C, extension 30 s 70 °C. Finally, 95 °C 10 s, 65 °C 60 s, and 97 °C 1 s were used to obtain the dissociation curves. The ramp rate was adjusted to 1 °C/s. Measurements were performed in a LightCycler96 (Roche Diagnostics, Switzerland) from three biological replicates per experimental group (F₁-γ and F₁-C). Linreg (v2017.0)⁶⁸ was used to determine primers efficiency and to calculate the expression level of miRNAs as measured by the threshold cycle values (Ct). Quantification values derived from five technical replicate per biological replicate. Relative quantification was normalized to a synthetic spike-in kanamycin RNA and expression levels were determined as equation 2^−ΔΔCT. Forward primers were designed with CLC-Workbench v6.0 (Qiagen, USA) (Table ​(Table88).

miRNA primers used for RT-qPCR validation (CLC-Workbench v6.0).

RT-qPCR values are given as log2 fold change (FC) of the expression in generation F₁ from exposed parents (F₁-γ) relative to generation F₁ from control parents (F₁-C). RNA-seq values are given as log2 FC and derived from differential expression analysis using edgeR (v3.24.3, Bioconductor) by pairwise comparison between F₁-γ and F₁-C group. Spearman’s correlation coefficient = 0.7667 (p = 0.02, confidence interval 95%, alfa < 0.05).

The obtained mean relative miRNA expression values (F₁-γ vs F₁-C) were compared to mean relative miRNA expression values for the same miRNAs from RNA-seq, and a Spearman’s correlation coefficient was calculated (p < 0.05) (Graphpad Prism 7 v7.0, La Jolla, USA).