4.5. Investigating the Efficacy of Vit-A and Vit-C Nanoemulsion on the In Vitro Expression of Milk-Specific Proteins

Mammary epithelial cells were seeded in a 12-well culture plate at a density of 2 × 10⁴ cells/well in DMEM (supplemented with 10% of FBS, 100 μg/mL of penicillin and streptomycin, 5 μg/mL of insulin, 50 μg/mL of gentamicin, and 1 μg/mL of hydrocortisone) medium added to the culture plates, and culture plates were kept in a humidified atmosphere, including 5% CO₂ at 37 °C. After 100% confluency, cells were treated with nanoencapsulated vitamins at a concentration of 100 mg/mL for 24 h. The experiment was conducted independently three times. The cellular RNA was isolated from MAC-T cells using the RNeasy lipid tissue kit (Qiagen, Valencia, CA, USA). The isolated RNA was quantified using a UVS-99 microvolume UV/Vis spectrometer (ACT gene, Piscataway, NJ, USA). The cDNA synthesis was carried out using 500 ng of cellular RNA with oligo (dT) primers and reverse transcriptase provided by the Superscript III first-strand synthesis system for RT-PCR (Invitrogen). The level of mRNA expression of milk proteins (casein) was assessed by SYBR Green-based real-time PCR on an ABI 7500 PCR system (Applied Biosystem, Foster City, CA, USA). The target gene expression levels were normalized with housekeeping genes. Primers used for qPCR are listed in Table 1.

Oligonucleotide primer sets for real-time PCR.