Sybr green pcr master mixture

The total RNA of the lungs or cells was isolated with triazole reagent (Thermo Fisher Scientific) based on the manufacturer's protocol. The cDNA was generated from the RNA samples using the Prime foot RTMasterMix (TAKARA Bio Inc., Shiga, Japan). Real-time PCR was carried out by the SYBR green PCR master mixture (Thermo Fisher Scientific) on a Thermal Cycle instrument (Jena Analysis, Germany). The specific primer sequences were shown in Table S2.

Total RNA was obtained from carotid artery by miRNeasy Mini Kit (NO.217004, QIAGEN, Germany) and reverse transcribed into cDNA using 5× All-In-One RT Mastermix (G490, Abm, China). RT-qPCR for specific genes was performed using SYBR Green PCR master mixture (Thermo Fisher) with custom-designed primers on the Roche LightCycler480 Real-Time PCR System. Results were normalized to GAPDH RNA, and the fold change was determined using the 2-ΔΔCT method. The following primers were used:
PDPN 5'GAGGCTCCAACGAGATCAAG-3' and 3'-CAGTAGCACCTGTGGTTGTT-5'.

The RevertAid First Strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) was used for the synthesis of cDNA from total RNA. To validate the transcriptomic data, real-time quantitative PCR was used to obtain an independent assessment of the expression of the mpd gene and the genes that integrate both PNP biodegradation pathways. The cDNA previously synthesized was used as a template for qRT-PCR experiments; the specific primers for each gene are presented in Table S1. Each reaction mixture contained five µl of SYBR green PCR master mixture (Thermo Scientific, Waltham, MA, USA), two µl of H₂O, forward and reverse primers in two µl, and the template in one μl. PCRs were performed with the Rotor-Gene Q (Qiagen, Hilden, Germany) using the following program: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The dissociation protocol was 95 °C for 15 s and 60 °C for 20 s, followed by a ramp from 60 to 95 °C for 20 min. The transcript of the recombinase A protein (recA) was used as an internal (unregulated) reference for relative quantification. The results of real-time RT-PCR were analyzed using the 2^−ΔΔC_T method (Livak & Schmittgen, 2001 (link)), and the data are expressed below as relative levels of expression. All reactions were done in triplicate (Table S1).