Mj opticon 2 machine

To test the overall validity of the expression levels of genes determined by DGE analysis, real time RT-PCR was performed. One microgram of total RNA from each sample was reverse transcribed using oligo-deoxythymidine as a primer in a 10 µl volume. Real time RT-PCR was performed using an MJ Opticon™⁻² machine (Bio-Rad, Hercules, CA). Actin (GenBank number: HO112155) and ubiquitin (GenBank number: HO112156) genes were used as internal controls. The reaction mixture (25 µl) contained 12.5 µl of SYBR Green Real-time PCR Master Mix (Toyobo), 0.5 µM of forward or reverse primers (Table 1 in File S1), and cDNA template (equivalent to 100 ng of total RNA). The amplification procedure was performed using the following parameters: 94°C for 30 s; 45 cycles at 94°C for 12 s, 58°C for 30 s, 72°C for 45 s and 80°C for 1 s for plate reading. A melting curve was generated for each sample to assess the purity of the amplified products. All experiments were carried out with three biological replicates. The expression levels were calculated from the threshold cycle using the delta-delta CT method.

To examine the reliability of the transcriptome results, six DEGs (fibronectin1 (fn1), integrinα5 (itga5), lysyl oxidase-like 2a (loxl2a), loxl2b, lectin galactoside-binding-like 1 (lgals1), and pleckstrin homology H1 (plekhh1)) and six DEGs (fn1, itga5, loxl2a, chemokine receptor 4 (cxcr4), vegfaa, and transforming growth factor-beta receptor 2 (tgfbr2)) in loach and yellow catfish were, respectively, selected to perform qPCR. Total RNAs isolated from the original and regenerated skins were reverse transcribed to first-strand cDNA using reverse transcriptase (Invitrogen, Waltham, MA, USA). The qPCR was carried out on an iQ5 system (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex Taq (TaKaRa, Dalian, China), according to the manufacturer’s instructions. The primers for these genes were designed manually (Table S1). The reaction mixture (10 μL) comprised 2.5 μL cDNA (1:4 dilution), 5 μL SYBR Premix Ex TaqTM II (TaKaRa), 0.5 μL specific forward primer (10 μM), 0.5 μL universal primer (10 μM), and 1.5 μL water. The reactions were performed in an MJ Opticon™-2 machine (Bio-Rad, Hercules, CA, USA). The relative expression levels were normalized to the endogenous control gene β-actin, and calculated by the 2^−ΔΔCt method [34 (link)].