Mir 200b

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.

Total RNA, including miRs, were extracted from the HAECs using a High Pure miRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). Variable miRs and RNU6B-specific cDNA were synthesized according to the TaqMan microRNA assay kit (RNU6B: Cat No. 602003; miR-200a: Cat No. P160705-000 H12; miR-141: Cat No. P160621-009 F07; miR-200b: Cat No. P160801-000 H20; miR-200c: Cat No. P160808-004 G12; miR-429: Cat No. P160720-008 G12) protocol (Applied Biosystems). RNU6B was used as an internal control. Quantitative real-time PCR was performed using a StepOnePlus Real-Time PCR instrument (Applied Biosystems).

For mRNA analysis, total RNA was extracted using the TRIzol (Life Techologies) reagent and retro-transcribed with the GoScript Reverse Transcription System (Promega) using oligo (dT) and random primers. qPCR analysis was performed with specific primers (Table 8) and analyzed as previously described [30 (link)]. qPCR analysis was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). Ribosomal protein L31 (rp-L31) was used as a reference gene to normalize quantitation of target genes for differences in the amount of total RNA in each sample.
microRNA levels were analyzed from total RNA using the following TaqMan MicroRNA Assays: miR-200a ID:000502, miR-200b ID:002251, miR-200c ID:002300 and RNU24 ID:001001(Applied Biosystems, Thermo Scientific). Relative expression was calculated using the comparative Ct method. The small nucleolar RNA U24 (RNU24) was used as a reference gene.
Both for mRNA and microRNA analysis, total RNA of HCC827 and HCC4006 parental cell lines were used as calibrator samples, relative to which differences in the RNA amount of resistant cell lines have been calculated. The data were analyzed using SDS (Ver. 1.4) software (Applied Biosystems).