Tb green premix ex taq 2 tli rnaseh plus 2x

Linseed oil (purity ≥99%) was purchased from Aladdin (Shanghai, China). Ciprofibrate (molecular weight = 289.15; purity >98%) was obtained from Aladdin (Shanghai, China). Enteric-coated aspirin was purchased from Bayer Healthcare Co. Ltd. (Beijing, China). Rat ELISA kits for 6-keto-prostaglandin F1α (6-keto-PGF1α), thromboxane B2 (TXB2), tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI) were acquired from RuiDaHengHui Science & Technology Development Co., Ltd. (Beijing, China). Human assay kits for fibrinogen (FIB) content were obtained from Mantino Medical Devices Co., Ltd. (Changchun, China). TRIzol (155960-18) was purchased from Ambin (Beijing, China). Triphenyltetrazolium chloride (TTC) Solution (2%) was purchased from Solarbio (Beijing, China). Red Blood Cell Lysis Buffer (C3702) and DEPC water (R0022) were provided by Beyotime (Shanghai, China). PrimeScript™ RT Master Mix (Perfect Real Time) (RR036A) and TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) (RR036A) were purchased from Takara (Beijing, China).

Gene expression analysis was performed by RT-PCR using Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Wilmington, DE, USA). Primers with Tm (melting temperature) 60 °C and 18–20 bp in length were designed by Primer 3 (Supplementary Table S2). IpEF1A was selected as the internal reference gene. PCR reaction mix (20 μl per well) contained 10 μl TB Green Premix Ex Taq II (TliRNaseH Plus) (2X) (Takara, Tokyo, Japan), 0.8 μl forward and reverse primers (10 μM) (Tsingke, Wuhan, China), 0.4 μl ROX Reference Dye II (Takara, Tokyo, Japan), 50 ng cDNA and RNA-free water. The two-step thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s. Corresponding gene expression level was analyzed with the 2-ΔΔCt method. Elongation factor 1-alpha was used as the internal control to normalize the relative amount of mRNAs for all samples.