Mir-X miRNA First-Strand Synthesis Protocol

The Mir-X miRNA First-Strand Synthesis kit (Clontech) was applied on total RNA (25 ng) or synthetic RNA (to obtain the miRNA standard curves as described for the miRCURY and miRPrimer strategies) according to the manufacturer’s instructions, in a final volume of 10 µL in the presence of mRQ enzyme. The reaction took place at 37 °C for 1 h. cDNA samples were diluted (40×) and 10 µL of PCR reaction including specific miRNA forward primer (the entire sequence of the miRNA isoform which included 4A) added to the 3′-end as previously described [14 (link)]; Table 1, the mRQ 3′ primer (Clontech) as reverse primer and 4.6 µL of diluted cDNA. Cycling conditions were identical to the ones used for miRPrimer2 strategy on the LightCycler^® 96 Instrument (Roche; Switzerland).