Platinum sybr green

Tissues of adult mice were harvested on the fifth day (ST) or three months (LT) after the last application and homogenized in TRIzol Reagent (Invitrogen) using a pestle. For RNA extraction, the TRIzol Reagent was used according to manufacturer’s recommendations. RNA was stored at -80 °C. RNA was reverse transcribed to cDNA using GoTaq Probe 2-Step RT-qPCR System (Promega) and cDNA was stored at -20 °C. qRT-PCR was performed via a StepOnePlus Real-Time PCR System (Applied Biosystems) using Platinum SYBR Green (Invitrogen). The following cycling conditions were used: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 sec, 60 °C for 1 min. Relative target gene expression was normalized to TBP. Primer sequences are listed in Table 1.

The total RNA was isolated by using TRIzol reagent (Invitrogen). The purity and quantity of RNA was analyzed by Nano drop spectrophotometer ND-1000 UV-Vis (NanoDrop Technologies, USA). The quantified RNA (500 ng) was used to synthesize cDNA by Thermal cycler (BIO-RAD, USA). The reaction mixtures (10 μl) were prepared using Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) according to the manufacturer instructions. The qPCR was performed in a 7300 real-time PCR system (Applied Biosystems, Warrington, UK) with platinum SYBR green (qPCR supermix uracil-DNA glycosylase with 6-carboxyl-X-rhodamine, Invitrogen). The total volume of reaction mixture was 10 μl, which contained 2.5 μl of cDNA, and 7.5 μl of master mix that included RT enzyme, SYBR green, forward and reverse primers (1 pmol/μl). The reaction cycles were performed first at 50°C for 5 min; followed by 95°C for 5 min; then 40 cycles at 95°C for 15 s, at 60°C for 30 s and at 72°C for 30 s. According to the minimum information for publication of qPCR experiments guidelines, β-actin was used as a housekeeping gene because of its high stability across porcine various tissues (27 (link), 28 (link)). Expression of β-actin was used to normalize cDNA levels for differences in total cDNA levels in the samples. Primers were described previously (11 (link), 13 (link)).

2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS), reduced glutathione (GSH), 5,5’dithio-bis-(2-nitrobenzoic acid) and nitroblue tetrazolium (NBT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Naringenin at 95% from Santa Cruz Biotechnology (Dallas, TX, USA). Tert-butyl hydroperoxide and 2-deoxy-D-ribose from Acros (Pittsburgh, PA, USA). Enzyme-linked immunosorbent assay (ELISA) kits from eBioscience (San Diego, CA, USA). Superscript® III, Oligo(dT)12-18 primers, Platinum SYBRGreen® and primers from Invitrogen (Carlsbad, CA, USA). Materials for formulations were from Galena (Campinas, SP, Brazil). All other reagents used were of pharmaceutical grade.

To validate the RNA sequencing analysis, 12 single gene patterns of expression through time were selected and verified with qRT-PCR. The genes were selected to represent various immune cells, chemokines and Vascular Endothelial Growth Factor A (VEGF), which have been shown to be involved in OTM. Their primer sequences are listed in the Appendix, Table 1. All oligonucleotides are shown in the 5′ to 3′ orientation. A total 750 ng-1 μg mRNA was reverse transcribed by using qScript cDNA Synthesis Kit (Quanta-BioSciences). Quantitative RT-PCR reactions (10 µl volume) were performed by using Platinum SYBR Green (Invitrogen), according to the manufacturer’s instructions. The qPCR data was analyzed by using QuantStudio 12 K software. The following reaction conditions were: 10 min at 95 °C, 45 cycles of 15 s at 95 °C, and 60 s at 60 °C. The samples were normalized to the GAPDH gene as control mRNA, by the change in cycling threshold (ΔCT) method and calculated based on 2 − ΔΔCT.

Cells were lysed from a 25–35 mm long section of ePTFE, and RNA was isolated from the lysate using an RNeasy mini isolation kit (Qiagen). Samples were reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturers' protocols. Gene expression was quantified using Platinum SYBR Green and ROX reference dye (Invitrogen) with in-house designed primers at 250 nM concentration (S1 Table). All in vitro samples were tested for a panel of coagulation and inflammation genes, specifically, CD39, EPCR, TM, eNOS, ICAM, VCAM, PECAM, TF, and TFPI. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene and ddct values were determined by comparing treated samples to static controls. Data were transformed by subtracting 1 from any positive 2^-ddct values and taking the negative inverse of any negative 2^-ddct values and adding 1. This resulted in a continuous, linear data set and is referred to as fold change, where the static values are 0. Positive values show an increase from paired static samples, while negative values indicate a decrease from the static control.

Total RNA was isolated from cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions and digested with DNase I (Qiagen) to remove genomic DNA. First strand cDNA synthesis was performed with Superscript III system (Invitrogen) using random primers and amplified using Platinum SYBR-Green (Invitrogen). For QPCR the Applied Biosystems 7900HT Fast Real time PCR or the Light Cycler 480 SYBR Green I Master Mix (Roche) systems were used. PCR primers were designed using Primer3 software. All experiments were performed in biological duplicates or triplicates for each time point analysed. Expression values were normalized against the β-actin or the TATA-binding protein (TBP) and standard deviations were calculated and plotted using Prism 6 software (GraphPad). Primer sequences are available upon request.

Total RNA was isolated from cells growing on matrigel using the RNeasy kit (QIAGEN) according to the manufacturer’s instructions and digested with DNase I (QIAGEN) to remove genomic DNA. First strand cDNA synthesis was performed with Superscript III system (Invitrogen) using random primers and amplified using Platinum SYBR-Green (Invitrogen). For qPCR the Applied Biosystems Quantstudio 6 Flex Real-Time PCR system was used. PCR primers were designed using NCBI Primer-Blast software, using exon-spanning junctions (Table S3). Expression values for each gene were normalized against GAPDH, using the delta CT method and standard deviations were calculated and plotted using GraphPad Prism 9. To compare gene expression data at different time points, the mean expression value at day 3 was calculated and used to determine the fold change for each gene at different time points. Error bars represent standard deviation across three biological replicate samples.