U6 snrna

Mature miR-770-5p levels were quantified with Qiagen miRNA assays. Quantitative PCR was performed using SYBR mix (Qiagen) on a Roche Light Cycler 480. U6 snRNA (MS00033740) was used as the internal control (Qiagen). Relative expression levels were calculated using the 2^-ΔCT method while fold changes were calculated using the equation 2^-ΔΔCT.

Complementary DNA (cDNA) was synthesized by reverse transcription of RNA using the miScript II RT Kit (Qiagen, Venlo, Netherlands). miRNA specific cDNA was created using HiSpec buffer, while mRNA specific cDNA was created using HiFlex buffer. qRT-PCR was performed using a Bio-Rad CFX96 Real-Time C1000 Touch Thermal Cycler. MiRNA cycling conditions were as follows: (1) 95°C for 15 min; (2) 40 cycles of 94°C for 15 sec, 55°C for 30 sec, 70°C for 30 sec (collection step). mRNA cycling conditions were similar, except for adjusted annealing temperatures on a primer-by-primer basis. miR-708-5p, U6 snRNA, miR-15a, COX-2, and mPGES-1 primers were purchased from Qiagen, while ODZ4 and GAPDH primers were purchased from Origene. Amplification was performed using the miScript SYBR Green PCR Kit (Qiagen). No template and no reverse transcriptase controls, as well as melt curve analysis, were implemented to ensure samples/primers were not contaminated. Quantitative Comparative C_T (ΔΔC_T) analysis was used to analyze gene expression changes relative to U6 snRNA/miR-15a (miRNA) or GAPDH (mRNA). qRT-PCR data represent the average of ≥ 3 biological replicates. Each sample was measured with n ≥ 2 technical replicates per target gene per independent experiment.

Complementary DNA (cDNA) was synthesized by reverse transcription of RNA using the miScript II RT Kit (Qiagen, Venlo, Netherlands). miRNA-specific cDNA was created using HiSpec buffer, while mRNA-specific cDNA was created using HiFlex buffer. qRT-PCR was performed using a Bio-Rad CFX96 Real-Time C1000 Touch Thermal Cycler. miRNA cycling conditions were as follows: (1) 95°C for 15 min, (2) 40 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s (collection step). mRNA cycling conditions were similar, except for adjusted annealing temperatures on a primer-by-primer basis. miR-708-5p, U6 snRNA, and COX-2 primers were purchased from Qiagen, while ODZ4, mPGES-1, and GAPDH primers were purchased from OriGene. Amplification was performed using the miScript SYBR Green PCR Kit (Qiagen). No template and no reverse transcriptase controls, as well as melt curve analysis, were implemented to ensure samples/primers were not contaminated. Quantitative comparative C_T (ΔΔC_T) analysis was used to analyze gene expression changes relative to U6 snRNA (miRNA) or GAPDH (mRNA). qRT-PCR data represent the average of ≥3 biological replicates. Each sample was measured with n ≥ 2 technical replicates per target gene per independent experiment.

RNA was extracted from keratinocytes and dermal fibroblasts using Quick-RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions. Extracted RNA was reverse transcribed using the First Strand cDNA Synthesis kit (ThermoFisher Scientific). The cDNA was then used in a qRT-PCR assay using QuantiFast SYBR Green PCR kit (Qiagen). For the assay, the following QuantiTech primers were used: IFNK (QT00197512; Qiagen), IFNB1 (QT00203763; Qiagen), IFNL1 (QT00222495; Qiagen), IFNA2 (QT00212527; Qiagen), U6snRNA (forward—5′-CTCGCTTCGGCAGCACA-3′; reverse—5′-AACGCTTCACGAATTTGC-3′; Sigma-Aldrich). For gene expression analysis, ddCt method was used and all samples were normalized to the housekeeping gene (U6snRNA).

Total miRNAs were prepared from ASCs and exosomes using a miRNeasy mini kit (Qiagen, Hilden, Germany). Total exosomes were prepared from ASC culture supernatant using total exosome isolation reagent (Thermo Fisher Scientific). RNA (250 ng) was reverse transcribed using a miRCURY LNA Universal RT microRNA PCR kit (Qiagen). Real-time PCR was performed using miR-150-specific primers (Qiagen). qRT-PCR was performed on a Step One Plus real-time PCR system (Life Technologies, Carlsbad, CA). After normalization to U6 snRNA (Qiagen), expression levels of each target miRNA were calculated using the comparative cycle threshold method.

Quantitative real-time PCR was used to measure the expression of mature miR-711. A unit of 100 ng of total RNA was reverse-transcribed using miScript II RT Kit (Qiagen). Reverse transcription reaction products (1 µl) were used for quantitative PCR (qPCR) with miScript SYBR Green PCR Kit (Qiagen) according to the manufacturer’s instructions. miScript Primer Assays for following miRs were used: rno-miR-711 (MS00017696); rno-miR-23a-3p (MS00033327); and U6 snRNA (MS00033740) (Qiagen).