Real-time RT-PCR analysis of melanogenic genes

The real-time RT-PCR analysis was performed using an ABI7900HT Instrument (Applied Biosystems, Waltham, MA, USA). For the TaqMan analysis, predesigned or optimized assays on demand (Applied Biosystems) were used, including MITF (ID: Hs01117294_m1), tyrosinase (ID: Hs00165976_m1), TRP-1 (ID: Hs00167051_m1), TRP-2 (ID: Hs01098278_m1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID: Hs00266705_g1), hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Hs02800695_m1), and 18S (Hs03003631_g1). The data were analyzed using ABI Sequence Detector Software version 2.0 (Applied Biosystems). Total RNA was extracted from cells using TRI reagent® according to the manufacturer's instructions and stored at -70°C until use. cDNA was synthesized from total RNA (1 μg) using MuLV reverse transcriptase according to the manufacturer's instructions. The real-time RT-PCR analysis was conducted as previously described [20 (link)]. The results were normalized to the expression level of endogenous GAPDH and were also tested against two additional housekeeping genes (18S and HPRT). We found that the results were not significantly different from those obtained using GAPDH. Expression levels of the target genes were normalized to the levels observed in the controls. The results were verified by repeating each experiment four times in triplicate.

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Hwang Y.S., Oh S.W., Park S.H., Lee J., Yoo J.A., Kwon K., Park S.J., Kim J., Yu E., Cho J.Y, & Lee J. (2019). Melanogenic Effects of Maclurin Are Mediated through the Activation of cAMP/PKA/CREB and p38 MAPK/CREB Signaling Pathways. Oxidative Medicine and Cellular Longevity, 2019, 9827519.

Publication 2019

ABI7900HT Instrument ABI Sequence Detector Software version 2.0

Abi 2 Assays Cdna Expression genes Glyceraldehyde 3 phosphate dehydrogenase Housekeeping genes Hypoxanthine guanine phosphoribosyltransferase Mitf Mulv Real time pcr analysis Reverse transcriptase Trp 1 Trp 2 Tyrosinase

Corresponding Organization : Sungkyunkwan University

Other organizations : Eulji University, Hongik University, Animal and Plant Quarantine Agency

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of MITF
Expression levels of tyrosinase
Expression levels of TRP-1
Expression levels of TRP-2

control variables

ABI7900HT Instrument (Applied Biosystems, Waltham, MA, USA) used for real-time RT-PCR analysis
TaqMan assays on demand (Applied Biosystems) used for analysis
GAPDH, HPRT, and 18S used as endogenous control genes
Total RNA extracted using TRI reagent® and stored at -70°C
CDNA synthesized from total RNA (1 μg) using MuLV reverse transcriptase

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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