Quantification of miR395 Expression

The reverse transcription was achieved using QuantiMir kit (System Biosciences, Mountain View, CA, USA), according to the manufacturer’s instructions, starting from 1 μg of total RNA. Quant Studio 12K Flex Real-Time PCR (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect the expression of miR395 with the following reaction mixture: 5 μL of Power Syber Green PCR master mix (Life Technologies, Carlsbad, CA, USA), 0.1 μL of forward primer, 0.1 μL of reverse primer, 1.4 μL of nuclease-free water, and 1 μL of each sample. The PCR program was set up with an initial denaturation at 95 °C for 10 min followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. The primer sequence used to detect miR395 was obtained from [20 (link),21 (link),22 (link),23 ,24 (link),25 (link),26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 ,38 (link)]. The threshold method was used to analyze miR395 expression and the ΔCt values, calculated based on Hajizadeh et al. [39 (link)], involved the use of 18S rRNA as an internal standard.