Gel documentation system

Total RNA was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA (2 µg) was reverse transcribed using oligo (dT)₁₆ primers to obtain cDNA. The cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Republic of Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). The amplified products were separated on a 2% agarose gel, stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA), and visualized with a gel documentation system (Fujifilm, Tokyo, Japan). The primer sequences were as follows: human parkin sense 5′-TCCTTCCTGCTGTCAGTGTG-3′, and antisense 5′-GCAGAGACCGTGGAGAAAAG-3′; human GAPDH sense 5′-GAAGGTGAAGGTCGGAGTC-3′, and antisense 5′-GAAGATGGTGATGGGATTTC-3′. Equal loading was verified based on the GAPDH level.

TRIzol (Invitrogen) was used to obtain total RNA extracts according to the manufacturer’s protocol. To synthesize cDNA, total RNA (2 μg) was reverse-transcribed using an oligo-dT₁₈ primer. The synthesized cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). PCR-amplified products were separated by 2% agarose gel electrophoresis and staining with ethidium bromide (Sigma, St. Louis) and imaged using a gel documentation system (Fujifilm). The following PCR primer sequences were used: human MMP3 sense 5′-AACCTGTCCCTCCAGAACCT-3′ and antisense 5′-GGAAGAGATGGCCAAAATGA-3′; human MMP9 sense 5′-CTCGAACTTTGACAGCGACA-3′ and antisense 5′-GCCATTCACGTCGTCCTTAT-3′; human Col1A1 sense 5′-CACAGAGGTTTCAGTGGTTTGG-3′ and antisense 5′-GCACCAGTAGCACCATCATTTC-3′; human GADD153/CHOP sense 5′-AGGGAGAACCAGGAAACGGAAACA-3′ and antisense 5′-TCCTGCTTGAGCCGTTCATTCTCT-3′; human and human GAPDH sense 5′-GAAGGTGAAGGTCGGAGTC-3′ and antisense 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH was used as a reference gene for normalization.

TRIzol (Invitrogen) was used to obtain the total RNA extract according to the manufacturer’s protocol. To synthesize cDNA, total RNA (2 µg) was reverse-transcribed using oligo dT18 primer. The synthesized cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). PCR-amplified products were separated using 2% agarose gel, including ethidium bromide (Sigma, St. Louis, MO, USA), and imaged in a gel documentation system (Fujifilm, Tokyo, Japan) [30 (link)]. The following primer sequences were used: mouse tyrosinase 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense) (NM_011661.5); mouse MITF, 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense) (NM_001178049.1); mouse TRP-1 5′-AGACGCTGCACTGCTGGTC AAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense) (NM_001282015.1). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR experiments [20 (link)].