Genemax thermal cycler

Total RNA was isolated from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and measured with a NanoDrop spectrophotometer (Tecan). mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qPCR). qPCR was conducted using SsoAdvanced Universal SYBR Green Supermix (BioRad) and the CFX Real-Time System (BioRad), and mRNA levels were normalized to those of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Relative expression was calculated using the 2^−ΔΔCT method. Semi-quantitative PCR was performed using 2X PCR MasterMix (Intron, Seoul, Korea) and the GeneMax thermal cycler (BIOER; Hangzhou, China) to visually identify the OR10A3 gene expressed in human embryonic kidney cells (HEK293T; ATCC). The primer sequences used for quantitative PCR are listed in Table 1.

The total RNA from the cells was isolated using TRIzol reagent (Invitrogen; Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, 1 µg total RNA was used to generate cDNA using oligo(dT) primer and SuperScript IV reverse transcriptase (Invitrogen). Quantitative PCR was performed with cDNA, primers listed in Supplementary Table S1, and SYBR green (BioRad) on a CFX Real-Time System (BioRad) to measure the OR, cell survival-, and collagen biosynthesis-related gene expression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The relative gene expression was quantified by the comparative “ct” (2^−ΔΔct) method. Semi-quantitative PCR was performed using a 2× PCR Master Mix (Intron; Seoul, Korea) and primers listed in Supplementary Table S1 on a GeneMax thermal cycler (BIOER; Hanqzhou, China) to detect the olfactory signaling pathway components in Hs68 cells. The PCR products were separated by electrophoresis on 1.2% agarose gels containing TopRed Nucleic Acid Gel Stain (Biopure; Horndean, UK) and were photographed under UV light using a CoreBio i-MAX Gel Image Analysis System (CoreBio system; Seoul, Korea).

Total RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified on a NanoDrop spectrophotometer (Tecan). Thereafter, an equal amount of each RNA sample was subjected to cDNA synthesis using SuperScript IV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. To visually identify the specific genes expressed in keratinocytes, semi-quantitative PCR was conducted in a 20 μL solution, comprising 2× PCR MasterMix (Intron, Seoul, Korea), 0.6 μM of each primer, and 25 ng template, using the GeneMax thermal cycler (BIOER; Hanqzhou, China). Negative controls containing mRNA without reverse transcription were used to confirm the absence of DNA contamination and PCR specificity. For precise quantification, quantitative PCR was carried out in a 20 μL solution, comprising 10 μL SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 0.6 μM of each primer, and 25 ng template cDNA, using the CFX Real-Time System (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the housekeeping gene for gene expression normalization. Primer sequences used for each gene are listed in Supplementary Table S1.