Sybr green real time pcr master mix

Total RNA was extracted with the TRIzol reagent following the standard instructions. The purified RNA was treated with DNase to remove DNA contamination and the first strand of cDNA was synthesized using a cDNA synthesis kit (11123ES60, Yeasen, China). The synthesized cDNA was used as a template for qRT-PCR using the SYBR-Green Real-Time PCR Master Mix (11202ES08, Yeasen, China). GAPDH was utilized as the internal reference gene for normalization of target gene expression. The RT-qPCR primers are listed in Table 1.17 (link)
Table 1

Primer Sequences

Name		Primer Sequences
Ki67	Forward	5’-AAACCCCACCAAGTAAAACA-3’
	Reverse	5’-CCAAGGCAAGCTCAGGAC-3’
GPX4	Forward	5’-AGTGGATGAAGATCCAACCCAAGG-3’
	Reverse	5’-GGGCCACACACTTGTGGAGCTAGA3’
PTGS2	Forward	5’-TGACCAGAGCAGGCAGATGA-3’
	Reverse	5’-CCAGTAGGCAGGAGAACATATAACA-3’
SLC7A11	Forward	5’-GTCTGGAGAAACAGCCAAGG-3’
	Reverse	5’-CGGAGTTCCTCGAATAGCTG-3’
ACSL4	Forward	5’-GTAATTGGTGGACAGAACATC-3’
	Reverse	5’-TACTCTCCTGCTTGTAACTTC-3’
GAPDH	Forward	5’-AAAGAAGCTCAACTACATGG-3’
	Reverse	5’-TGCAAAGAATGCGTCCCAGAG-3’

Total RNA from the murine skin biopsies and RAW264.7 cells was isolated and purified by using the RNAsimple total RNA kits (Tiangen). Subsequently, total RNA (500 ng per unit) was reversely transcribed by the Hifair^TM 1st Strand cDNA Synthesis SuperMix regents (Yeasen, Shanghai, China) with consecutive incubation of 25 °C for 5 min, 42 °C for 30 min. and 85 °C for 5 min. Real-time quantitative PCR analyses were conducted with the SYBR® Green Realtime PCR Master Mix regents (Yeasen) according to the directions on the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster city, CA, USA) for PCR amplification with holding stage (95 °C for 30 s), cycling stage (95 °C for 15 s and 60 °C for 34 s for 40 cycles), and melt curve stage using the gene-specific primers in Supplementary Table S3 by the ΔΔ Ct method.

The graft samples were placed at −80°C for temporary storage. Total RNA was
extracted from graft and cell samples by RNAiso Plus (Takara Biomedical
Technology, Dalian, China). Then the stable cDNA was obtained by using the Hieff
First Strand cDNA Synthesis Super Mix (Yeasen, Shanghai, China). To accurately
assess the mRNA expression, the qRT-PCR with SYBR Green real-time PCR Master Mix
(Yeasen, Shanghai, China) was used. The β-actin was used as the internal
reference. Primer sequences were shown below:
CD206: Forward Primer 5′- CTCTGTTCAGCTATTGGACGC-3′;

Reverse Primer 5′-TGGCACTCCCAAACATAATTTGA-3′;

Arg-1: Forward Primer 5′-CTCCAAGCCAAAGTCCTTAGAG-3′;

Reverse Primer 5′- GGAGCTGTCATTAGGGACATCA-3′;

Fizz-1: Forward Primer 5′- CCAATCCAGCTAACTATCCCTCC-3′;

Reverse Primer 5′-ACCCAGTAGCAGTCATCCCA;

PPARγ: Forward Primer 5′-GGAAGACCACTCGCATTCCTT-3′;

Reverse Primer 5′- GTAATCAGCAACCATTGGGTCA-3′;

Fabp4: Forward Primer 5′-AAGGTGAAGAGCATCATAACCCT-3′;

Reverse Primer 5′- TCACGCCTTTCATAACACATTCC-3′;

C/EBP-α: Forward Primer 5′-CAAGAACAGCAACGAGTACCG-3′;

Reverse Primer 5′-GTCACTGGTCAACTCCAGCAC-3′;

Adipoq: Forward Primer 5′-TGTTCCTCTTAATCCTGCCCA-3′

Reverse Primer 5′-CCAACCTGCACAAGTTCCCTT-3′;

β-actin: Forward Primer 5′-GGCTGTATTCCCCTCCATCG-3′

Reverse Primer 5′-CCAGTTGGTAACAATGCCATGT-3′

RNA was extracted using TRIzol reagent (Invitrogen, USA). cDNA was synthesized using prime Script RT kit (TaKaRa, Japan). qRT-PCR was performed with SYBR Green Realtime PCR Master Mix (Yeasen, Shanghai, China) using the following PCR primers: RNF128, fwd: 5ʹ-CTGCTCGAAGGCTACGGAATG-3′ and rvs: 5ʹ-GTGTGCGTAGTTGAAGCCTTCC-3ʹ. GAPDH, fwd: 5ʹ-GTCTCCTCTGACTTCAACAGCG-3ʹ and rvs: 5ʹ-ACCACCCTGTTGCTGTAGCCAA-3ʹ.