Iq sybr green mix

Hindpaw skins of MCI-4W mice were collected 3 h after the intraplantar injection. Total RNA was extracted using Direct-zol™ RNA MiniPrep Kit (Zymo Research Corporation) and 0.5–1 µg of RNA was reverse-transcribed using the iScript cDNA Synthesis® (Bio-Rad). Specific primers including GAPDH control were designed using IDT SciTools Real-Time PCR software. We performed gene-specific mRNA analyses using the MiniOpticon Real-Time PCR system (BioRad)^{35 (link)}. Quantitative PCR amplification reactions contained the same amount of Reverse transcription (RT) product, including 7.5 µL of 2× iQSYBR-green mix (BioRad) and 100–300 nM of forward and reverse primers in a final volume of 15 µL. The primer sequences were shown in Supplementary Fig. 12b. Primer efficiency was obtained from the standard curve and integrated for calculation of the relative gene expression, which was based on real-time PCR threshold values of different transcripts and groups.

Liver samples from mice were flash frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Livers were homogenized in Lysing Matrix D tubes (MP Biomedicals) containing 700 μl RLT buffer with 1% (v/v) β-mercaptoethanol (QIAGEN) using a FastPrep-24 (MP Biomedicals) bead beater for 45 s at 6.5 m/s (repeated 3 times, incubating on ice for 1 minute in between rounds). Homogenates were centrifuged at 20,000 × g at 4°C for 5 minutes to pellet debris, RNA was extracted using phenol:chloroform:IAA, pH 6.7 (Sigma), and samples were mixed with 50% ethanol prior to being transferred to QIAGEN RNeasy columns, according to the manufacturer’s instructions. Cleaned RNA samples were subjected to DNase treatment (Invitrogen) prior to cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad). RNA was removed through the addition of 1 N NaOH at 65°C for 30 minutes, followed by the addition of an equal volume of 1 N HCl. cDNA was cleaned using a PCR clean-up kit (Promega) according to the manufacturer’s instructions. cDNA concentrations were adjusted to 1 ng/μl using the Synergy 2 with Gen 5 software (Bio-Tek) prior to qRT-PCR using iQ SYBR Green mix (Bio-Rad), gene-specific primers (Table S1), and a CFX96 qPCR cycler (Bio-Rad). Transcript abundance was calculated using the ΔΔCt method and data were normalized to β-actin gene expression.