Total RNA was extracted using TRIzol reagent (Thermo Fisher SCIENTIFIC), and cDNA was synthesized from one µg of RNA using random primers and iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). mRNA levels were quantified using IQ SYBR green supermix (Bio-Rad Laboratories) on the CFX96 real-time system instrument (Bio-Rad). Specific primers located in different exons of the same gene were designed to detect relative mRNA levels. The housekeeping β-2 microglobulin or c-ABL genes were used for internal normalization. All the PCR reactions were performed in triplicate, and PCR products were also visualized on agarose gels after ethidium bromide staining. The oligonucleotide sequences are reported in Supplemental Table S2. Relative fold variations were calculated using the 2−ΔΔCt method by the formula: 2−(sampleΔCt − controlΔCt), where ΔCt is the difference between the amplification fluorescent thresholds of the gene of interest and the internal reference gene/s used for normalization [39 (link)].
To detect the levels of mature miR-128, total RNA was prepared with TRIzol reagent (Thermo Fisher SCIENTIFIC) and miR amounts were evaluated by using the TaqMan miRNA assay kit (Thermo Fisher SCIENTIFIC). For normalization of RNA levels, the amounts of small nucleolar RNA RNU6 (Thermo Fisher SCIENTIFIC) were measured [39 (link)].
To detect the levels of mature miR-128, total RNA was prepared with TRIzol reagent (Thermo Fisher SCIENTIFIC) and miR amounts were evaluated by using the TaqMan miRNA assay kit (Thermo Fisher SCIENTIFIC). For normalization of RNA levels, the amounts of small nucleolar RNA RNU6 (Thermo Fisher SCIENTIFIC) were measured [39 (link)].