Mir 222

Total RNA extraction and purification from milk, as well as specific miRNA analysis, were performed as previously described [19 (link)]. The small RNA-specific RT and PCR primers used were U6 snRNA (Assay ID: 001973), miR-26a (Assay ID: 000405), miR-27a (Assay ID: 000408), miR-29a (Assay ID: 002112), miR-103 (Assay ID: 000439), miR-200a (Assay ID: 000502), miR-200b (Assay ID: 001800), miR-221 (Assay ID: 000524), and miR-222 (Assay ID: 002276) (Applied Biosystems). The threshold cycle (Ct) was calculated by the instrument’s software (StepOne Software v2.2.2, Applied Biosystems) and miRNA expression was calculated as a percentage of the control group at day 5 of lactation using the 2^−ΔΔCt method [61 (link)]. Expression of U6 snRNA and β-actin were used as references. Finally, miRNA expression was calculated according to each reference and data are presented as the average of both values. The primers used for the β-actin gene are detailed in Table S1 and were supplied by Sigma.

The miRNA-7 and miR-221/222 expression levels were analyzed by a quantitative real-time PCR, using plasma samples. After genotyping, 49 individuals were randomly chosen among the patients (n = 22) and the healthy individuals (n = 27) and plasma miRNAs were isolated using the commercial kit GRS microRNA Kit (GRISP) according to the manufacturer’s instructions. MicroRNA samples were then used as a template for cDNA synthesis using TaqManMicroRNA Reverse Transcription Kit (Applied Biosystems). The circulating miRNAs expression levels were analysed by a quantitative real-time PCR. Reactions were carried out on a StepOne One qPCR machine, containing 1x Master Mix (Applied Biosystems), with 1x probes (TaqMan MicroRNA Assays miR-7: 002314, miR221: 002096, miR-222: 002097, Applied Biosystems), cDNA sample, and the RNU48 endogenous control (TaqMan MicroRNA Assay: 001006, Applied Biosystems) was used to normalize the results, regarding the two biomarkers, since it presents a constant expression level. The data analysis was carried out using the StepOne Software v2.2 (Applied Biosystems) with the same baseline and threshold set for each plate, in order to generate threshold cycle (Ct) values for all the genes in each sample.