Total cellular RNA was extracted using the RNeasy Kit (Qiagen). To prepare the cDNA, 1 µg cellular mRNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). PCR amplification was conducted using the StepOnePlus™ Real-Time PCR System in reaction mixtures (20 µL) containing SYBR® Green PCR Master Mix, 60 ng cDNA, and 2 pmol gene-specific primer sets (Macrogen, Seoul, Korea). The primers used for PCR analysis are as follows: MMP1 (GenBank accession numbers NM_001145938.1) 5'-CAT ATA TGG ACG TTC CCA AAA TCC-3' (forward) and 5'-GTG CGC ATG TAG AAT CTG TCT TTA A-3' (reverse); SFN (NM_006142.3) 5'-AGA GAC ACA GAG TCC GGC AT-3' (forward) and 5'-ATG TCC TCA TAG CGT TCG GC-3' (reverse); glyceraldehyde 3-phosphate dehydrogenase (GAPDH; NM_0020-46.3) 5'-ATG GGG AAG GTG AAG GTC G-3' (forward) and 5'-GGG GTC ATT GAT GGC AAC AA-3' (reverse). Reactions were performed under the following conditions: 50℃ for 2 min, 95℃ for 10 min, 40 amplification cycles (95℃ for 15 s and 60℃ for 1 min), followed by a dissociation step. Melting curve analysis showed single peaks, which supported the homogeneity of amplicons. The mRNA expression level relative to the internal control GAPDH was calculated using the comparative threshold cycle method.
Gene specific primer sets
Manufactured by Macrogen
Gene-specific primer sets are laboratory equipment used for the amplification of specific gene sequences during PCR (Polymerase Chain Reaction) experiments. These primers are designed to target and replicate defined regions within a given gene or genome.
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2 protocols using gene specific primer sets
1
Quantification of MMP1 and SFN mRNA
2
Quantification of Melanogenic Gene Expression
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Cellular mRNA was extracted using the RNeasy kit (Qiagen, Valias, CA, USA), and complementary DNA (cDNA) was synthesized using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). The qRT-PCR analysis was performed with the StepOnePlus™ real-time PCR system (Applied Biosystems). The reaction mixture (20 µL) consisted of SYBR® Green PCR Master Mix (Applied Biosystems), cDNA (60 ng), and gene-specific primer sets (2 pmol) (Macrogen, Seoul, Korea). The primer sequences are shown in Table 2 .
Thermal cycling parameters for PCR were set as follows: initial incubation at 50 °C for 2 min, DNA polymerase activation at 95 °C for 10 min, 40 amplification cycles (annealing and extension at 60 °C for 1 min and melting at 95 °C for 15 s). Melting curves were generated to verify the homogeneity of the amplified products. The mRNA levels of TYR, TYRP1, and DCT were normalized to that of an internal standard, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the relative Ct method. Ct is defined as the number of cycles required for the PCR signal to exceed the threshold. Fold changes in the test group compared to the control group were calculated as 2−ΔΔCt, where ΔΔCt = ΔCt(test) − ΔCt(control) = [Ct(gene, test) − Ct(reference, test)] − [Ct(gene, control) − Ct(reference, control)].
Thermal cycling parameters for PCR were set as follows: initial incubation at 50 °C for 2 min, DNA polymerase activation at 95 °C for 10 min, 40 amplification cycles (annealing and extension at 60 °C for 1 min and melting at 95 °C for 15 s). Melting curves were generated to verify the homogeneity of the amplified products. The mRNA levels of TYR, TYRP1, and DCT were normalized to that of an internal standard, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the relative Ct method. Ct is defined as the number of cycles required for the PCR signal to exceed the threshold. Fold changes in the test group compared to the control group were calculated as 2−ΔΔCt, where ΔΔCt = ΔCt(test) − ΔCt(control) = [Ct(gene, test) − Ct(reference, test)] − [Ct(gene, control) − Ct(reference, control)].
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