We isolated miRNA and RNA from cultured cells with TriPure reagent (Sigma-Aldrich).
For miRNA quantification, 1 μg total RNA was reverse transcribed by using the NCode™ VILO™ miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). We amplified 10 ng of total RNA equivalents with iQSyber Green Supermix (Bio-Rad Laboratories, Temse, Belgium) using commercial miRNA-specific forward primers (Qiagen) and a reverse universal primer (provided in the NCode VILO miRNA cDNA Synthesis Kit).
For mRNA quantification, 2 μg of total RNA was reverse transcribed as described previously [11 (link), 39 (link)], and qPCR was performed with designed primers (Additional file3 : Table S1).
The threshold cycles (Ct) were measured in separate tubes and in duplicate. To ensure the quality of the measurements, each plate included a negative control for each set of primers, and analysis of the melting curve was carried out at the end of the amplification. Cyclophilin (mouse) and TATA-box-binding protein (TBP, human) were used as reporter genes. Relative changes in the expression level of one specific gene were presented as 2-ΔΔCt [11 (link), 39 (link)].
For miRNA quantification, 1 μg total RNA was reverse transcribed by using the NCode™ VILO™ miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). We amplified 10 ng of total RNA equivalents with iQSyber Green Supermix (Bio-Rad Laboratories, Temse, Belgium) using commercial miRNA-specific forward primers (Qiagen) and a reverse universal primer (provided in the NCode VILO miRNA cDNA Synthesis Kit).
For mRNA quantification, 2 μg of total RNA was reverse transcribed as described previously [11 (link), 39 (link)], and qPCR was performed with designed primers (Additional file
The threshold cycles (Ct) were measured in separate tubes and in duplicate. To ensure the quality of the measurements, each plate included a negative control for each set of primers, and analysis of the melting curve was carried out at the end of the amplification. Cyclophilin (mouse) and TATA-box-binding protein (TBP, human) were used as reporter genes. Relative changes in the expression level of one specific gene were presented as 2-ΔΔCt [11 (link), 39 (link)].
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