The total RNA was extracted from the skin using a single-phase solution of phenol and guanidine isothiocyanate (311-02501; ISOGEN, Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. A region containing PC was collected from the edge of the wound under a microscope. The collected total RNA was mixed with PrimeScript IV 1st strand complementary DNA (cDNA) Synthesis Mix (6215A, Takara Bio, Shiga, Japan), and was promptly reverse transcribed into cDNA.
Cdna synthesis mix
Manufactured by Takara Bio
Sourced in Japan, China
The CDNA Synthesis Mix is a reagent kit designed for the reverse transcription of RNA into complementary DNA (cDNA). It contains all the necessary components, including reverse transcriptase, to efficiently convert RNA samples into cDNA that can be used in downstream applications such as PCR and qPCR.
Automatically generated - may contain errors
3 protocols using cdna synthesis mix
1
Skin RNA Extraction and cDNA Synthesis
2
RNA Extraction and Quantitative PCR
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Total RNA was extracted from frozen tissues using TRIzol reagent according to the manufacturer's instructions. The purities and concentrations of total RNA samples were determined by NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE) . 200 ng of total RNA was reverse transcribed using cDNA Synthesis Mix (Takara, Dalian, China). Real-time PCR was carried out in an Applied Biosystems QuantStudio 5 Real-Time PCR System (ABI, Warrington, UK) with SYBR Green PCR master mix (Takara, Dalian, China) and gene-specific primers. The sequences for the forward and reverse primers used to quantify mRNA are listed in Supplementary Table 1. The following conditions were used for real-time PCR: 95°C for 10 min, then 95°C for 15 sec, and 60°C for 1 min in 40 cycles. The 2 -ΔΔCT method (Pfaffl, 2001) (Bustin et al., 2009) .
3
qPCR Analysis of Target Genes
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qPCR was performed to determine the mRNA expression of target gene, TF, and PAI-1. Total RNA in RLE-6TN was extracted by using TRIzol reagent (Invitrogen), followed by cDNA synthesis, where the cDNA synthesis mix was purchased from Takara, and cDNA was diluted and amplified according to the manufacturer’s instructions and was measured by using a qPCR system. The relative expression of the target gene was calculated by the 2−ΔΔCt method. Amplified transcript levels for each specific gene was normalized to GAPDH. Primers were provided by Sacred Engineering, see Additional file 2 :Table S3.
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