Quantitative Analysis of miRNA Expression

Expression of miR-17, miR-21, miR-18a and let-7g in B16 cells was analyzed using stem-loop PCR technology^{43 ,44 (link)}. cDNA synthesis was carried out using SuperScript III reverse transcriptase (SSIII RT, Invitrogen, USA) as previously described^{45 (link)}. The RT and PCR primers used in the study are presented in Table S1 (Supplementary information). PCR amplification was carried out in a total volume of 20 µl, using Maxima Hot Start Taq DNA polymerase (Thermo Scientific, USA), 1 × PCR Buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 1 × EvaGreen (Biotium, Hayward, USA), and 0.2 mM of PCR sense and antisense primers. The reaction was performed with initial preheating at 94 °C for 4 min and 40 cycles of 94 °C for 40 s, 61 °C for 30 s, and 72 °C for 30 s, followed by melting point determination. The obtained PCR data were analyzed using standard Bio-Rad iQ5 v.2.0 software. For each sample, the threshold cycle (Ct) was determined. Quantitative assessment of the level of transcripts representation and relative miRNA expression was performed by comparing the Ct values for miR-17 and a reference U6.