Quantitative Analysis of miRNA Expression

Each reaction was performed with 5 µL of 2× TaqMan^® Fast Advanced Master Mix (Applied Biosystems^®) with 0.5 µL of 20× TaqMan^® MicroRNA Expression Assays (miR-466l-3p: 002804; miR-466k: 240990_mat; miR-223-3p: 002295; miR-125b-5p: 000449; snoRNA-202: 001232; Applied Biosystems^®), 3 µL of nuclease-free water and 1.5 µL of cDNA sample, making a total volume of 10 µL. The quantification was performed in duplicate, and CT standard deviation values superior to 0.5 were excluded. Negative controls lacking cDNA were also included in all reactions. All target and endogenous controls for each sample were amplified in the same plate. The thermal cycling conditions for all assays were the following: 20 s at 95 °C followed by 45 cycles of 1 s at 95 °C and 20 s at 60 °C. The same baseline and threshold were set for each plate using the analysis software for qPCR from the Thermo Fisher Connect platform (Thermo Fisher Scientific, Waltham, MA, USA), in order to generate threshold cycle (Ct) values for all of the miRs/SnoR in each sample. Small nucleolar RNA 202 (snoR-202) was previously tested by our group in this mouse model, was the one that showed the lowest standard deviation values in chest and ear, and therefore, was used as endogenous control [14 (link),15 (link)].