Fast start universal sybr green master rox

Eight EAT tissues and SAT tissues were collected from Xiangya Hospital. This study was approved by the Ethical Committee of Xiangya Hospital and conducted in accordance with the Declaration of Helsinki. In addition, each patient volunteered written informed consent. All tissues were immediately frozen in liquid nitrogen after resection and stored in liquid nitrogen. The clinical characteristics of included CAD patients were listed in Table 1.
Total RNA was extracted from tissues using TRIzol (Takara, Japan) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a cDNA Synthesis Kit (Takara, China). mRNA levels were tested using Nanodrop one (Thermo Fisher Scientific, Waltham, MA, USA). All reactions were performed on the Eppendorf Mastercycler ep realplex (2S; Eppendorf, Hamburg, Germany) using following cycling parameters: 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 34 s. RT-qPCR was performed using the FastStart Universal SYBR^® Green Master (ROX) (Takara, China). The primer sequences were listed in Data S1.
The relative expression level for each target gene was normalised by the Ct value of β-actin (internal control) using a 2^−ΔΔCt relative quantification method. A meaningful analysis between the two groups was performed by a paired t-test, and a P value < 0.05 was considered statistically significant.

Total RNA was extracted from the samples using TRIzol (Takara) according to the manufacturer’s instruction. We used the cDNA synthesis kit (Takara) to conduct reverse transcription. Real-time quantitative PCR (RT-qPCR) was performed by the FastStart Universal SYBR Green Master (ROX) (Takara). 2^–ΔΔCt relative quantification method was used for relative expression. β-actin was used as the internal reference (Sangon Biotech). The primer sequences were as following: UCP1 forward CGACGTCCAGTGTTATTAGGTA, UCP1 reverse GTAGAGTTTCATCCGCCCTTC, GNG3 forward CGGTGAACAGCACTATGAGTAT, GNG3 reverse TCACAGTA AGTCATCAGGTCTG, MCHR1 forward CGCTTGGTCC TGTCGGTGAAG, MCHR1 reverse GCCTTTGCTTTCTG TCCTCTCCTC, BDKRB1 forward CTTTTTGTCCTGTTGG TCTTCC, BDKRB1 reverse CTGATGAACAAATTGGCCTTGA, MCHR2 forward AACCTGGCTGTGGCTGATTTGG, MCHR2 reverse GGGATGTGATGATGGTGCAGAGAG, CXCL8 forward AACTGAGAGTGATTGAGAGTGG, CXCL8 reverse ATGA ATTCTCAGCCCTCTTCAA, CXCR5 forward CGGCAG ACACGCAGTTCCAC, CXCR5 reverse ACGGCAAAGGGCAA GATGAAGAC, CCR8 forward TGGCCCTGTCTGACCT GCTTT, CCR8 reverse GGCATAAGTCAGCTGTTGGCT, CCL4L1 forward CTCAGTATCAGCACAGGACACAGC, CCL 4L1 reverse AGAGACAGGACAGTCACGCAGAG, TAS2R10 forward AGTGTTTGGGGTTTTGGGGAATGG, TAS2R10 reverse AGCCGGTGAGAATAAAGCCAATCG, TAS2R41 forward ACTCAGCCACCTTCTGGTTTTGC, and TAS2R41 reverse ATCAGGACAGAGCCCAACAGGAG.