2.14. Transfection of EL4 cells with mature miR-30a and anti-miR-30a and determination of expression of miR-30a and Becn1 in the presence of DES or VEH

Freshly cultured EL4 cells (5 × 10⁶) were transfected with mature miR-30a and anti-miR-30a using Lipofectamine RNAMAX transfection kit from Invitrogen and following the protocol (Reverse Transfection) of the company (Invitrogen), as described previously (Hegde et al., 2013; Singh et al., 2015a,b). We also used p-GFP as a positive control for transfection. Forty-eight hrs post transfection, EL4 cells were treated with VEH or DES (10 μM/ml) for 24 h. The expression of miR-30a and Becn1 in EL4 was determined post-treatment with DES or VEH. In brief, total RNAs including miRs from EL4 cells transfected with miR-30a, anti-miR-30a, or miR-30a and anti-miR-30a and treated with DES or VEH were isolated using RNeasy mini kit from QIAGEN and following the protocol of the company (QIAGEN, Valencia, CA). First strand cDNA synthesis was performed on total RNA (1 μg) using iScript cDNA Synthesis Kit and following the protocol of the company (Bio-Rad). Expression of miR-30a and Becn1 was determined performing Real-Time PCR. Snord96a primer pairs were used as an internal control for miR-30a and 18S primer pairs were used as an internal control for Becn1 gene. Western blotting was also performed to determine the expression of Becn1 in transfected EL4 cells.