Perfecta sybr green fastmix

qPCR was used to detect the expression of vasculogenesis-associated genes (Supplementary Table 3). Candidate primers and the reference beta-2 microglobulin (β2M) genes were designed using the Primer-Blast database (NCBI, USA). For qPCR reactions, synthesized cDNA (1 μl) was used in a 20 μl reaction containing PerfeCTa® SYBR Green FastMix (10 μl, Quanta Biosciences, USA) and sense and antisense primers (300 nм) using a MasterCycler Realplex4 (Eppendorf, Germany). Reaction conditions comprised an initial 2 min denaturation step at 95 °C, followed by 45 cycles of 95 °C denaturations for 10 s, a 30 s annealing step at the required temperature (Supplementary Table 3), and a 10 s elongation step at 72 °C. Transcripts abundances were normalized to the expression of β2M. Samples were run in triplicate in each assay. Normalized expression values were calculated following the mathematical model proposed by Pfaffl using the formula: 2^{−ΔΔCt 54 (link)}.

Total RNA was prepared using TRIzol (Thermo Fisher Scientific, Cat#:15596018) according to the manufacturer's protocol. cDNA was synthesized using the qScript™ cDNA synthesis kit (Quanta Biosciences, Cat#:95049). QPCR analysis was performed using gene-specific primers, PerfeCTa SYBR® Green Fast Mix (Quanta Biosciences, Cat#95047), and Roche Lightcycler apparatus and software. Oligonucleotide primers were designed using PrimerBLAST (NCBI) and listed in Supplementary Table (ST) 1, 18 s was used as housekeeping.

RNA from E8.5 or E9.5 embryos (respectively, 200 and 500 ng) was reverse‐transcribed using the qScript cDNA synthesis kit (Quanta Biosciences). A 1/10 dilution of the cDNA was used to perform qPCR on a Bio‐Rad iCycler with PerfeCTa SYBR Green FastMix (Quanta Biosciences). Primer pairs used can be found in Appendix Table S2. qPCRs for each embryo were run in triplicate, and the average of these technical replicates was taken to represent one independent experiment. Comparisons of such data from at least three embryos (see Figure legends) were made for each condition and each gene of interest. All data were determined using ΔΔCt method 107 and shown relative to GAPDH with error bars indicating ±SEM. A one‐way ANOVA with a Tukey post‐test was performed for statistical analyses (see original source data).

Real-time PCR (CFX96 Real Time System; Bio-Rad, Hercules, CA, USA) was carried out with the PerfeCTa SYBR Green FastMix (Quanta Biosciences). Species-specific primer sequences for gyrB of P. aeruginosa were designed using primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) using P. aeruginosa and B. cenocepacia sequences obtained from GenBank. The forward primer and the reverse primer were 5′-GGTGTTCGAGGTGGTGGATA-3′ and 5′-TGGTGATGCTGATTTCGCTG-3′, respectively. The specificity of the primers was evaluated by melting curve analysis.
To generate a standard curve, DNA extracted from serially-diluted and PMA-treated planktonic P. aeruginosa cultures was used for qPCR. The C_q-values obtained were plotted against the number of viable cells determined by SPC. The serial dilutions were prepared from a P. aeruginosa overnight suspension (OD 0.1). Cells were diluted from 10⁹ CFU/ml to 10⁴ CFU/ml in PS. Six independent biological repeats were included.

RNA was extracted from tissues and HUVEC using the RNeasy kit (Qiagen). Supercript III Reverse Transcriptase (Invitrogen) was used for first strand cDNA synthesis. Quantitative real-time PCR was carried out using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system. Oligonucleotides used for human and mouse are listed in Supplementary Tables 3, 4, respectively.