Table 6. Fold Change (FC) comparison from HTS differential expression study and RT-qPCR data.
| HTS | RT-qPCR | |||||||
| miRNA/Cluster | EU2 vs. EA3 | EU vs. AS4 | EA vs. AS | ANOVA group factor p-value | EU vs. EA | EU vs. AS | EA vs. AS | ANOVA group factor p-value |
| Hsa-miR-200b-3p | −1.45 | −1.54 | −1.06 | 0.794 | 1.29 | 1.50* | 1.16 | 0.012 |
| Ssc-miR-126 | −1.07 | 1.24 | 1.32 | 0.576 | 1.09 | 1.60*** | 1.47*** | <.001 |
| Ssc-miR-99a | 1.63 | 3.05 | 1.87 | 0.237 | 1.10 | 1.29** | 1.18§ | 0.005 |
| Hsa-miR-200c-3p | −2.31 | −1.73 | 1.34 | 0.266 | 1.39 | −1.15 | −1.61** | 0.009 |
| Ssc-miR-126* | −1.36 | −1.18 | 1.15 | 0.352 | 1.08 | 1.35§ | 1.25 | 0.044 |
| Ssc-miR-92a | −1.87 | −1.68 | 1.11 | 0.168 | 1.20 | 1.02 | −1.18 | 0.614 |
| Ssc-miR-26a | −2.15 | −1.29 | 1.67 | 0.235 | 1.13 | 1.14 | 1.01 | 0.602 |
| Bta-miR-193b | 2.23 | 5.46 | 2.45 | 0.285 | 1.25 | 1.55** | 1.25 | 0.003 |
| Ssc-miR-532-5p | −1.16 | −2.49 | −2.14 | 0.082 | 1.05 | −1.06 | −1.12 | 0.685 |
| Ssc-miR-423-5p | 5.28 | 3.76 | −1.40 | 0.110 | 1.09 | 1.14 | 1.05 | 0.416 |
| Ssc-miR-29c | −3.78 | 1.45 | 5.47 | 0.028 | 1.01 | 1.20 | 1.19 | 0.105 |
| Ssc-miR-486 | −5.66 | −1.55 | 3.64 | 0.179 | −1.48* | −1.28 | 1.16 | 0.025 |
| Ssc-let-7f | −2.76 | −11.60 | −4.21 | 0.344 | 1.22 | 1.33* | 1.09 | 0.017 |
| Cl-2 (Hsa-miR-31-3p)1 | - | - | - | - | 1.05 | 1.12 | 1.07 | 0.631 |
| Cl-5 (Hsa-miR-194-5p)1 | - | - | - | - | 1.02 | 1.24 | 1.22§ | 0.049 |
| Cl-25 (Hsa-miR-138-5p)1 | - | - | - | - | 1.35** | 1.36** | 1.00 | 0.003 |
| Cl-29 | - | - | - | - | −1.03 | 1.21 | 1.24* | 0.009 |
| Cl-38 (Bta-miR-1468)1 | - | - | - | - | 1.03 | 1.60*** | 1.55*** | <.001 |
: miRBase (v18) homology (e-value≤1e-03). Internal mismatches were accepted.
: EU: European breeds. 3: EA: European commercial breeds. 4: AS: Asian breeds.
For HTS data, fold changes from sequence counting between breed groups were calculated from normalised data in counts per thousand for each library and averaged per groups. Positive and negative signs indicate that the level of gene expression is higher for the first or the second group of the test, respectively. Analysis of variance (ANOVA) including breed as fixed factor was performed. Significance was set at P<0.05. Fold changes for clusters could not be calculated due to their low total counts.
For qPCR data expression study, the quantity obtained of each miRNA in each sample was normalised by the Normalization Factor and corrected in relation to the lowest normalised value. Analysis of variance (ANOVA) including the RT and breed as fixed factors was performed and fold change from Least Squares Means (LSM) between breed groups were calculated. Significance was set at P<0.05. Scheffe test determined whether there was significant differential expression between breed groups (§: suggestive p-value<0.1, *: significant with p-value<0.05, **: significant with p-value<0.01, ***: significant with p-value<0.001).