Rna isolation reagent

Total RNAs were extracted from different tissues using RNA Isolation Reagent (Invitrogen, Carlsbad, CA) and reversely transcribed to produce cDNA (Fermentas, EU). Relative expression of messenger RNA (mRNA) species was determined by Real Time PCR with Faststart Universal SYBR Green PCR Master (Roche Diagnostics, USA) by ABI7300. The primer sequences were shown in Additional file 1: Table S1. Relative mRNA levels were normalized with β-actin, and results were expressed as fold amplification.

In 0.5 mL TRIzol reagent (RNA Isolation Reagent, Invitrogen—ThermoFisher Products & Kits, Amresco, LLC-Solon, Waltham, MA, USA), 50 mg of dorsal skin tissue was homogenized using an ultrasonic homogenizer (Sonics-Vibracell, Sonics and Materials Inc., Newtown, Fairfield County, CT, USA). According to the guidelines of the producer, total RNA was extracted from dorsal skin tissues, and the concentration of RNA yield and purity were calculated [48 (link)].

Total RNA of cells was extracted using RNA isolation reagent (Invitrogen, USA) and then was reverse-transcribed using One Step RT-qPCR Kit (Sangon Biotech, China) according to the manufacturer's protocol. Next, equal amounts of cDNA were used for RT-qPCR. PCR reaction and real-time detection were performed using iQ5 Real-time Quantitative PCR (Bio-Rad, USA). As shown in Table 1, the appropriate forward and reverse real-time PCR primers were used for GAPDH, VEGF, Bcl-2, BAX, LC3B, Caspase 3, mTOR, and Beclin-1. The real-time PCR cycles included predenaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 20 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. 2^−ΔΔCt (ΔΔCt = (C_mRNA–Ct_GAPDH) − (control − Ct_GAPDH)) was used to quantify the relative expression of target mRNA.