Nanodrop spectrophotometer

To examine the relative expression levels of miRNA and mRNAs, total RNA was extracted from the skin tissues or cultured cells with TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. A NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.) was used to record the concentration and quality of RNA. On assessing the RNA quality and concentration using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.), equal volumes of RNA samples (3.0 µg) were collected from each group for cDNA library synthesis with the Bestar™ qPCR RT kit (cat. no. 2220; DBI Bioscience), according to the manufacturer's protocol, at 37°C for 1 min, 50°C for 60 min and 70°C for 15 min. Then, relative levels of miRNA or mRNA were measured by RT-qPCR using the Bestar™ qPCR MasterMix kit (cat. no. 2043; DBI Bioscience), according to the following procedure: Pre-denaturation at 95°C for 2 min, with 42 cycles of denaturation at 94°C for 20 sec; annealing at 58°C for 20 sec and extension at 72°C for 20 sec. The maximum cycle number considered for gene expression is 42. U6 and GAPDH were used as the internal standard for quantitation of miRNA and EPHA7 expression, respectively. Expression levels were assessed by the standard 2^−ΔΔCq method (22 (link)). The experiment was repeated three times. The primers used for RT-qPCR are listed in Table II.

For double-stranded RNA (dsRNA) synthesis, a 423 bp fragment of the target gene (CYP6AE10) was selected from the open reading frame (ORF), a 688 bp fragment from green fluorescent protein (GFP) (GenBank accession ACY56286). Both target and control gene was first amplified by PCR. The primers used for the CYP6AE10 amplifications were designed to add the T7 polymerase promoter sequence at the 5 ends. Two pairs of primers (CYP6AE10-F and T7CYP6AE10-R, T7CYP6AE10-F and CYP6AE10-R) were used to amplify CYP6AE10 (Table 2). As a control, dsGFP was synthesized using the same method by two pairs of primers (GFP-F and T7GFP-R, T7GFP-F and GFP-R) (Table 2). PCR products were purified using the QIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA), and DNA concentrations were determined using a NanoDrop® spectrophotometer (Thermo Fisher, MA, USA). The dsRNA corresponding to CYP6AE10 was prepared from the purified PCR-generated templates according to the instructions provided by the commercially available kit (T7 RiboMAXTM Express RNAi System (Promega). After preparation dsRNA product was purified by MEGA clear (Ambion) and the resulted dsRNAs integrity were quantified by NanoDrop® spectrophotometer (Thermo Fisher, MA, USA) and stored at − 80 °C prior to use.