Platinum sybr green qpcr supermix udg

Total RNA was extracted from dissected aortic valve leaflets (from 4- and 10-month old mice) using RNeasy mini-kit (Qiagen) and reverse transcription was performed using the Moloney murine leukemia virus reverse transcriptase (ThermoFisher) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using 2X Platinum SYBR Green qPCRSuperMix-UDG (ThermoFisher) and MX3005p qPCR System cycler (Stratagene) according to the manufacturer’s protocol. Primers of target genes were designed using the Genscript PCR primer design tool (Table S3). ΔΔCT were determined with MxPro v4.10 software using cyclophilin A as a housekeeping gene.

Total RNA was extracted by Trizol® reagent and converted to complementary DNA (cDNA) with TransScript first-strand cDNA synthesis supermix kit (TransGen) on a thermocycler following the manufacturer’s protocol. QPCR analysis of scd-1, ICAM-1, VCAM-1, IL-6 and gadph were performed in an ABI 7900HT QPCR system using Platinum SYBR Green QPCR SuperMix-UDG with ROX. The reaction mixture was subject to the thermal cycling program as follows: heating up to 95 °C in 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 1 min^{59 (link)}. The expressions of target genes were normalized with that of GAPDH.

Quantitative PCR was performed using a Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, 11733046) on an ABI ViiA7 real-time PCR system (Applied Biosystems) following the manufacturer’s protocol. Each quantitative PCR well contained 1.25 ng cDNA together with the Platinum SYBR Green qPCR SuperMix-UDG, ROX reference dye (ABI, 7500) and a primer mix of the forward and reverse primers (0.25 μM each). Primer sequences are provided in Supplementary Table 12.

For both mRNA and miRNA quantification, total RNA was extracted using the TRIZOL Reagent (Invitrogen). Five micrograms of total RNA was used to synthesize the first-strand cDNA with M-MLV (Invitrogen). Real-time PCR was amplified in 20 μl reaction mixtures using the following parameters: 95°C for 1 min, followed by 40 cycles of 95°C for 20 seconds and 56°C for 40 seconds. The 20 μl qPCR reaction consisted of 2X Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, Life Technologies, Grand Island, NY, USA, 11733-046), 10 ng cDNA and 0.4 mM of forward and reverse primers. β-actin was used as the internal control. The specificity of the PCR reaction was monitored by a melt–curve protocol. For both mRNA and miRNA quantification, the data were imported into qBasePLUS (Biogazelle, Zwijnaarde, Belgium) for analysis of reference gene quality control and relative quantification. The primers used in this study are shown in Table 2.

Total RNA was extracted from frozen ventricles using RNeasy mini-kit (Qiagen) and reverse transcription was performed on 1 µg RNA using the Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using 2X Platinum SYBR Green qPCRSuperMix-UDG (Invitrogen) and MX3005p qPCR System cycler (Stratagene) according to manufacturer’s protocol. Primers of target genes were designed using the Clone Manager software and were the following: NOX4-Fwd: 5′-ATC​CTT​TTA​CCT​ATG​TGC​CGG-3′; NOX4-Rev: 5′-CTT​TCT​GGG​ATC​CTC​ATT​CTG​G-3′; NOX2-Fwd: 5′-AGT​GCC​CAG​TAC​CAA​AGT​TC-3′; NOX2-Rev: 5′-GTC​CCA​CCT​CCA​TCT​TGA​ATC-3′; Cyclophilin A-Fwd: 5′-CCG​ATG​ACG​AGC​CCT​TGG-3′; Cyclophilin A-Rev: 5′-GCC​GCC​AGT​GCC​ATT​ATG-3′. ΔΔCT were determined with MxPro v4.10 software using cyclophilin A as a housekeeping gene.

RNA was extracted with PeqGOLD TriFast (PEQLAB Biotechnologie). cDNA was synthesised from 1 µg RNA using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time RT-PCR was performed with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies) on a StepOnePlus Real Time PCR System (Applied Biosystems). Relative mRNA expression was calculated using the 2^−ΔΔC(t) method with Rn18s as an internal control. For direct comparison, mRNA expression of Slc2a1 and Slc2a4 was primer efficiency corrected as described previously⁶¹ . The primer sequences used are as follows:
Acta2, forward 5′-CAGGCATGGATGGCATCAATCAC-3′, reverse 5′-ACTCTAGCTGTGAAGTCAGTGTCG-3′; Col1a1, forward 5′-CCCTGGTCCCTCTGGAAATG-3′, reverse 5′-GGACCTTTGCCCCCTTCTTT-3′; Has1, forward 5′-TATGCTACCAAGTATACCTCG-3′, reverse 5′-TCTCGGAAGTAAGATTTGGAC-3; Has2, forward 5′-CAAAAATGGGGTGGAAAGAG-3′, reverse 5′-ACAGATGAGGCAGGGTCAAG-3′; Has3, forward 5′-CTCAGTGGACTACATCCAGG-3′, reverse 5′- GACATCTCCTCCAACACCTC-3′; Slc2a1, forward 5′-GCATCTTCGAGAAGGCAGGT-3′, reverse 5′-GTCCAGCTCGCTCTACAACA-3′; Slc2a4, forward 5′-ATCATCCGGAACCTGGAGG-3′, reverse 5′-CGGTCAGGCGCTTTAGACTC-3′; Pfkm, forward 5′-GGGACACCATCAGCCTTTGA-3′, reverse 5′-TCCCCTCCAAAAGTGCCATC-3′; Gys1, forward 5′-CGAATCCCTTTTAGTGGGGAG-3′, reverse 5′-TGAGGAGTCGTCCAGCATGT-3′; Rn18s, forward 5′-GCAATTATTCCCCATGAACG-3′, reverse 5′-GGCCTCACTAAACCATCCAA-3′.

Total RNA was isolated using the ToTALLY RNA kit (Ambion) and quantified with a Qubit fluorometer (Life Technologies) unless otherwise stated. Reverse transcription for qRT-PCR analysis was performed using High Capacity cDNA Reverse Transcription Kit (Life Technologies). qRT-PCR was performed on a QuantStudio 6 (Life Technologies) and/or 7900HT (Applied) with Taqman probes (Life Technologies) or Platinum SYBR Green qPCR supermix-UDG (Life Technologies) and gene-specific primers. Primers and Taqman probes used for qRT-PCR are listed in S3 Table.