Steponeplus rt pcr system

The cDNA (n = 5 for each group) was reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol for RT-PCR. RT-PCR was performed using the PowerUp SYBR Green PCR Master Mix (Thermo Fisher Scientific Inc.) and StepOnePlus RT-PCR System (Thermo Fisher Scientific Inc.). The primer sequences were determined using the Primer-Basic Local Alignment Search Tool (National Center for Biotechnology Information, Bethesda, MD, USA) (Table 1). The cycling parameters were as follows: 50 cycles of PCR (denaturing for 15 s at 95°C, annealing for 15 s at 60°C, and extension for 1 min at 72°C) after thermal activation for 2 min at 50°C and 95°C. Multiple housekeeping genes were used as internal standards. The ratio of mRNA expression of each target to that of peptidylprolyl isomerase A (Ppia), S100 protein, beta polypeptide, neural (S100b), and hypoxanthine guanine phosphoribosyl transferase (Hprt) was compared among the groups [28 (link), 29 (link)].

Cells at 80% confluence were treated with 2 μg/mL actinomycin-D (MCE) and collected at the indicated time points. Total RNA extracted by TRIzol Kit (Omega, Norcross, GA, USA) was quantified was performed using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) produced with the cDNA synthesis kit (Takara, Otsu, Japan) was quantified by qPCR with SYBR Green PCR Master Mix (Yishen, Shanghai, China) in a StepOnePlus RT-PCR system (Thermo Fisher Scientific). Primers for RT-qPCR were listed in Table S5.

The ICR mice’s skin was injected or treated with siRNA targeting scramble siRNA or siFFaR2 followed by topical application or treatment with fermented media of S. epidermidis with CIN 2% or BA within 24 h in mouse skin or 3T3 cells. The total RNA was isolated using the Purelink RNA mini kit (Invitrogen, USA) and homogenized to reduce the viscosity of difficult tissue samples (Invitrogen, USA). RNA (100 ng) was converted into complementary deoxyribonucleic acid (cDNA) using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). All sets were designed using the National Center for Biotechnology Information (NCBI) Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primerblast/). The reaction was performed on StepOnePlus RT- PCR System (ThermoFisher) using Power SYBRGreen PCR Master Mix (Thermo Fisher Scientific). The reaction conditions for 40 cycles are as follows: 95 °C for 10 min followed by 95 °C for 15s, 55 °C for 60 s, and 72 °C for 30 s. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used for normalization. The levels of relative expression levels were calculated using the cycle threshold (2-∆∆Ct) method [70 (link),71 (link)]. The primers used for FFAR2 and GAPDH were 5′-ACCCAAGAGCAGCTGGATGT-3′ (forward); 5′-AGCGCCTAACAGAAGATGGT-3′ (reverse) and 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward); 5′-GATGCCTGCTTCACCACCTT-3′ (reverse), respectively.

Total RNA was separated using the manufacturer’s directions by the Gene JET RNA Purification Kit. Revert Aid H—Reverse Transcriptase was used to backward-transcribe 5 g of total RNA into cDNA. Utilizing the cDNA as a framework, the CNTF gene’s relative expression was discovered utilizing the Applied Biosystem, Step One Plus RT-PCR system (Thermo Fisher Scientific, SA, Australia).
The primers were created using software (Primer 5.0), and they had the following sequences that were unique to rats (Table 6):
Thermo Fisher Scientific, SA, Australia (Catalog# K0221) supplied 12.5 μL of the qPCR Master Mix (2X Maxima SYBR Green/ROX), to which 2 μL of cDNA material, 1 μL of reverse primer, 1 μL of forward primer, and 8.5 μL of nuclease-free water were added to create a PCR mix of 25 μL. Thermal cycling occurred under the following circumstances:
Initial DNA denaturation was conducted for 10 min at 95 °C. This was followed by DNA-denaturation amplification (40 cycles) for 15 s each, 30 s of annealing at 60 °C, and 30 s of extension at 72 °C. For analysis of the melting curve, the temperature was raised from 63 to 95 °C upon the completion of the previous cycle. The comparative cycle threshold (Ct) technique was used to assess the relative amounts of gene expression, which were then normalized to the housekeeping gene [67 (link)].