M mlv reverse transcriptase

For RT-qPCR, total RNA was extracted from the eWAT and liver by using TRIzol reagent (GeneAll Biotechnology, Seoul, Republic of Korea) [20 (link)]. cDNA was synthesized with M-MLV reverse transcriptase (Bioneer, Daejeon, Republic of Korea) by using an mRNA and cDNA synthesis kit with poly (A) polymerase tailing (ABM Inc., Richmond, BC, Canada) for miR. Next, RT-qPCR was performed with a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany) by adding Greenstar qPCR Master Mix (Bioneer). Table S2 lists the primer sequences designed by Primer3 [24 (link)], and mRNA expression was normalized using β-actin as a reference control. The specific primers of miR-34a, miR-370, and RNU6 were purchased from ABM Inc., and the expression levels of miRs were normalized using the expression of RNU6 snRNA as a control. The mRNA and miR expression levels were calculated via the 2^−∆∆Ct method for relative quantification [25 (link)]. They were then expressed as fold differences compared with those of the HF group.

RT-qPCR was measured as previously described [19 (link)]. Total RNA from the 3T3-L1 adipocytes was extracted using TRIzol reagent (GeneAll Biotechnology, Seoul, Korea), and the cDNA was synthesized from RNA extracted using M-MLV reverse transcriptase (Bioneer, Daejeon, Korea). Afterward, RT-qPCR was performed in a fluorescent thermal cycler (Rotor-Gene^TM 3000, Corbett Research, Mortlake, NSW, Australia) using Universal SYBR^® Green PCR Master Mix (Bioneer Co., Daejeon, Korea). The 2^−ΔΔCt method was used as a relative quantification strategy, and target gene expression was normalized using β-actin as an endogenous control. Primers used for RT-qPCR analysis are shown in Table 1.

B16F10 melanoma cells (5 × 10⁴ cells/mL) were treated with the indicated concentrations of GABA (0–20 mM) in the presence or absence of 500 ng/mL α-MSH for 48 h. Total RNA was extracted using an easy-BLUE RNA Extraction Kit (iNtRON Biotechnology, Sungnam, Korea), and cDNA was synthesized using MMLV reverse transcriptase (Bioneer, Daejeon, Korea). MITF, tyrosinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was evaluated by amplifying the cDNA using EzWay PCR Ready Mix (KOMA Biotechnology) with specific primers (Table 1) [44 (link)].

To compare mRNA levels of cell cycle regulators and proteasome subunits, first their cDNA was obtained by reverse transcription with oligo-dT and M-MLV reverse transcriptase (Bioneer). Real-time quantitative PCR (RT-qPCR) was then performed with CFX96 Real-Time System (Bio-Rad) by using iTaq Universal SYBR Green Supermix (Bio-Rad). The RT-qPCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s, and melt curve stage were 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The RT-qPCR was performed with three biological replications, and relative transcript levels of gene of interest were calculated according to the 2^−ΔΔCt method against β-actin mRNA. Primer sequences used for the RT-qPCR are provided in Supplemental Table 2.

Total RNA was purified using TRIzol reagent (Thermo Fisher Scientific, Pittsburgh, PA) from the cerebral cortex of mice (n = 10). RNA was reverse transcribed to cDNA using MMLV reverse transcriptase (Bioneer, Daejeon, South Korea) and an oligo-d(T)18 primer. Quantitative real time PCR (qRT-PCR) was carried out using Rotor Gene SYBR Green supermix Kit (QIAgen, Hilden, Germany) and fluorescence was measured using Rotor-gene PCR Cycler (QIAgen, Hilden, Germany). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The primers were synthesized by Bioneer. The sequences for forward and reverse Syn primers were as follows: Syn I F: 5´- CAGGGTCAAGGCCGCCAGTC-3´and R: 5´-CACATCCTGGCTGGGTTTCTG-3´; Syn II F: 5´-AGGGGAGAAATTCCCAC-3´ and R: 5´-CCCAGAGCTTGTACCG-3´; Syn III F: 5´-CCAACAG-CGACTCTCG-3´ and R: 5´-GGTTGCGGATTGTCTC-3´ [20 (link)]. GAPDH primer was purchased from QIAgen. Three biologically independent experiments were performed and each PCR reaction was carried out in triplicate. The relative levels of specific mRNA were calculated by normalizing to the expression of GAPDH using the 2^-ΔΔCt method. Additionally, the expression values of the RF-EMF exposed groups were normalized to those of the sham-exposed group.

Total RNA in the head kidney was extracted using the Trizol^® method (Life Technologies™, Carlsbad, CA, USA). The isolated RNA was treated with DNase I at a final concentration of 200-unit/mL (Sigma) and quantified using a NanoVue^plus (Biochrom US Inc., Holliston, MA, USA). One microgram of total RNA was used for synthesizing cDNA using M-MLV Reverse Transcriptase (Bioneer, Korea), and real-time PCR was performed to evaluate the mRNA expression level of cytokines, such as interleukin-1β (IL-1β), interleukin-10 (IL-10), chemokine-1 (CK-1), and tumor necrosis factor-α (TNF-α) [22 (link)], toll-like receptor (TLR)2, cluster of differentiation 36 (CD36), serum amyloid A (SAA), cluster of differentiation 3 (CD3), heat shock protein 70 (HSP70), and secreted type of IgM (sIgM). The expression of elongation factor 1 (Ef-1α) was used as an internal control, and fold-changes were calculated using the 2^–ΔΔCt method.