2.7. miRNA Target Validation by RT-qPCR

RNA samples used for SOLID sequencing were also employed for miRNA target validation by RT-qPCR analysis. Total RNA was reverse transcribed to complementary DNA (cDNA) with RetroScript Kit (Ambion, Carlsbad, CA, USA) following manufacturer instructions with 50 ng/μL oligo (dT)15 (Promega, Madison, WI, USA) and 2.5 μM random hexamers (Applied Biosystems) and amplification was carried out on a StepOnePlus Real-Time PCR system (Applied Biosystems) thermocycler, using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The cycling parameters were: initial polymerase activation step at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing and elongation at 60 °C for 1 min. For each sample, three biological replicates (with 3 technical replicates each) were analyzed and RPS18 gene (ribosomal protein S18, Gene ID: 107882131) was used to normalize gene expression.

LinRegPCR software [22] was employed for the analysis of RT-qPCR experiments and data were analyzed by Student’s t-test for statistically significant differences (p < 0.05).

The list of RT-qPCR primers for miRNA target genes amplification can be found in Additional file 1, Table S1.